Recombinant humanized XVII type collagen as well as preparation method and application thereof

Abstract

The invention provides recombinant humanized XVII type collagen as well as a preparation method and application thereof, and relates to the technical field of biology. The recombinant humanized XVII type collagen provided by the invention has 100% sequence identity with human collagen, is good in water solubility, high in thermal stability, not easy to degrade, good in cell adhesion, migration and proliferation activity, capable of promoting vascular endothelial cell tube forming ability, free of hemolytic reaction, free of sensitization during subcutaneous injection, free of rejection risk and good in biocompatibility; the compound has the effects of promoting skin barrier injury repair induced by ultraviolet radiation and inhibiting oxygen free radical and inflammatory factor increase induced by ultraviolet radiation, has the activity of promoting wound tissue repair, collagen regeneration and hair follicle regeneration, and can be applied to the industrial fields of medical instruments or cosmetics and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant humanized type XVII collagen and its preparation method and application. Background Art

[0002] Type XVII collagen is a transmembrane protein expressed by epidermal basal keratinocytes. As a structural component of the hemidesmosome structure in the dermal-epidermal basement membrane zone of the skin, type XVII collagen plays an important role in the physiological functions of keratinocytes. The absence and / or dysfunction of type XVII collagen can lead to the detachment of keratinocytes from the basement membrane, and then lead to the separation of the epidermis and the dermis (Reference: Theranostics. 2022 Sep; 12(15): 6446-6454.).

[0003] Re-epithelialization is an important link in skin wound healing. Keratinocytes migrate to the skin injury area for repair, and then restore the skin barrier function. Research shows that cells with high expression of type XVII collagen exhibit stronger cell renewal ability, which helps to replenish lost cells during the re-epithelialization process (Reference: Nature. 2019 Apr;568(7752):344-350.). During the wound healing process, type XVII collagen promotes the activation, adhesion, migration and proliferation of keratinocytes, and then plays an important role in the processes of wound re-epithelialization, healing and skin barrier repair.

[0004] Type XVII collagen consists of an intracellular domain of 560 amino acids, a transmembrane domain of 23 amino acids and an extracellular domain of 914 amino acids. The extracellular domain contains alternately distributed collagen domains and non-collagen domains. Sixteen non-collagen domains divide its extracellular domain into 15 collagen domains. The longest of these collagen domains is COL15, which consists of 242 amino acids (residues 567 to 808), and the lengths of other collagen domains vary from 14 to 45 amino acid residues. Previous studies have reported that COL15 is the cell adhesion domain of type XVII collagen. After the extracellular domain of type XVII collagen is released to the extracellular matrix by proteolysis on the cell surface, COL15 can mediate cell adhesion and migration. (Reference: J BiolChem. 2001 Oct ;276(42):38673-9.).

[0005] Animal-derived type XVII collagen is extremely difficult to extract. Recombinant humanized type XVII collagen has a lower cost and is suitable for large-scale production, and has the advantages of high biocompatibility, low immunogenicity, good water solubility, and no pathogen biosafety hazards. In recent years, recombinant humanized type XVII collagen has been widely used in the fields of functional skin care products, medical dressings, etc.

[0006] In view of this, the present invention is hereby provided. Summary of the Invention

[0007] The first object of the present invention is to provide a recombinant humanized type XVII collagen to solve the above problems.

[0008] The second object of the present invention is to provide a nucleic acid molecule.

[0009] The third object of the present invention is to provide a vector.

[0010] The fourth object of the present invention is to provide a cell.

[0011] The fifth object of the present invention is to provide a method for preparing the above-mentioned recombinant humanized type XVII collagen.

[0012] The sixth object of the present invention is to provide the application of the above-mentioned recombinant humanized type XVII collagen in medical devices or cosmetics.

[0013] The seventh object of the present invention is to provide the application of the above-mentioned recombinant humanized type XVII collagen in a culture medium.

[0014] In order to achieve the above objects, the following technical solutions are specifically adopted: In the first aspect, the present invention provides a recombinant humanized type XVII collagen, which comprises a basic repeating unit, and the amino acid sequence of the basic repeating unit is as shown in SEQ ID NO.1; The number of the basic repeating units is at least 1. When the number of the basic repeating units is greater than or equal to 2, adjacent basic repeating units are connected by peptide bonds.

[0015] As a further technical solution, the number of the basic repeating units is 3; The amino acid sequence of the recombinant humanized type XVII collagen is as shown in SEQ ID NO.2.

[0016] In the second aspect, the present invention provides a nucleic acid molecule, which encodes the above-mentioned recombinant humanized type XVII collagen.

[0017] As a further technical solution, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.3.

[0018] In the third aspect, the present invention provides a vector, which carries the above-mentioned nucleic acid molecule.

[0019] Fourth aspect, the present invention provides a cell, which carries the nucleic acid molecule described above, or contains the vector described above, or expresses the recombinant humanized type XVII collagen described above.

[0020] As a further technical solution, the cell includes Escherichia coli.

[0021] Fifth aspect, the present invention provides a method for preparing recombinant humanized type XVII collagen, including: expressing the recombinant humanized type XVII collagen by using the cell described above, and then preparing the recombinant humanized type XVII collagen after separation and purification.

[0022] Sixth aspect, the present invention provides the application of recombinant humanized type XVII collagen in medical devices or cosmetics.

[0023] Seventh aspect, the present invention provides the application of recombinant humanized type XVII collagen in culture media.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The recombinant humanized type XVII collagen provided by the present invention has 100% sequence identity with human collagen, has good water solubility, high thermal stability, is not easily degraded, has good activities of cell adhesion, migration and proliferation, promotes the ability of vascular endothelial cells to form tubes, has no hemolytic reaction, has no sensitization after subcutaneous injection, has no risk of rejection, and has good biocompatibility; it has the effects of promoting the repair of skin barrier damage induced by ultraviolet radiation and inhibiting the increase of oxygen free radicals and inflammatory factors induced by ultraviolet radiation, has the activities of promoting wound tissue repair, collagen regeneration and hair follicle regeneration, and can be applied to industries such as medical devices, cosmetics or culture media. Description of the Drawings

[0025] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0026] Figure 1 : Plasmid map of recombinant humanized type XVII collagen PSQ12x3; Figure 2 : SDS-PAGE detection map of the expression and purification of recombinant humanized type XVII collagen PSQ12x3; Figure 3 : SDS-PAGE detection map of the digestion products of recombinant humanized type XVII collagen PSQ12x3; Figure 4 : SDS-PAGE detection map of the recombinant humanized type XVII collagen PSQ12x3 enzyme-digested product passing through a Ni column to remove the tagged protein; Figure 5 : SDS-PAGE detection map of the recombinant humanized type XVII collagen PSQ12x3 repeatedly passing through a Q column to remove endotoxin; Figure 6 : The recombinant humanized type XVII collagen PSQ12x3 promotes the adhesion of human epidermal keratinocyte cell line HaCaT; Figure 7 : The recombinant humanized type XVII collagen PSQ12x3 promotes the migration of human epidermal keratinocyte cell line HaCaT; Figure 8 : The recombinant humanized type XVII collagen PSQ12x3 promotes the proliferation of human epidermal keratinocyte cell line HaCaT; Figure 9 : Biosafety evaluation of the recombinant humanized type XVII collagen PSQ12x3 without hemolytic effect; Figure 10 : Biosafety evaluation of the recombinant humanized type XVII collagen PSQ12x3 without sensitization after subcutaneous injection; Figure 11 : Effect of the recombinant humanized type XVII collagen PSQ12x3 on the tube formation ability of vascular endothelial cells HUVEC; Figure 12 : Repair effect of the recombinant humanized type XVII collagen PSQ12x3 on UV radiation-induced skin barrier damage; Figure 13 : The recombinant humanized type XVII collagen PSQ12x3 inhibits the increase of oxygen free radicals induced by UV radiation; Figure 14 : Evaluation of the antioxidant, anti-inflammatory and pro-angiogenic effects of the recombinant humanized type XVII collagen PSQ12x3 on UV radiation-induced HUVEC damage; Figure 15 : H&E staining and MASSON staining of the recombinant humanized type XVII collagen PSQ12x3 promoting the repair of mouse skin wound tissue and the repaired tissue; Figure 16 : The recombinant humanized type XVII collagen promotes the proliferation of human hair follicle dermal papilla cells (HFDPC); Figure 17 : Immunofluorescence staining (keratin 15 and β-catenin) of the recombinant humanized type XVII collagen promoting hair follicle regeneration in mouse skin wound tissue. Detailed implementation methods

[0027] The embodiments of the present invention will be described in detail below in conjunction with embodiments and examples. However, those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. Those not specified in specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through commercial purchase.

[0028] The term "vector" refers to a nucleic acid vehicle into which a nucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted nucleotide, the vector is called an expression vector. The vector can be introduced into the host cell by transformation, transduction or transfection, so that the genetic element carried by it can be expressed in the host cell.

[0029] The vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses. In some embodiments, the vectors of the present invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and polyU sequences, etc.).

[0030] The expressions "cell", "cell line" and "cell culture" used herein are used interchangeably and all such names include progeny. The progeny may not be exactly the same as the primary cells due to natural, accidental or intentional mutations, for example, there are differences in morphology and / or genomic DNA from the primary cells. "Transformant" and "transformed cell" include the primary test cells and the cultures derived therefrom.

[0031] In a first aspect, the present invention provides a recombinant humanized type XVII collagen, the recombinant humanized type XVII collagen includes a basic repeating unit (denoted as PSQ12), and the amino acid sequence of the basic repeating unit is shown in SEQ ID NO.1: GSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAK (SEQ ID NO.1).

[0032] The number of the basic repeating units is at least 1. When the number of the basic repeating units is greater than or equal to 2, adjacent basic repeating units are connected by peptide bonds.

[0033] Through the research of the inventors, it is found that the recombinant humanized type XVII collagen provided by the present invention has good water solubility, high thermal stability, is not easily degraded, has good activities of cell adhesion, migration and proliferation, promotes the ability of vascular endothelial cell tube formation, has no hemolytic reaction, has no sensitization after subcutaneous injection, has no risk of rejection, and has good biocompatibility; it has the effects of promoting the repair of skin barrier damage induced by ultraviolet radiation and inhibiting the increase of oxygen free radicals and inflammatory factors induced by ultraviolet radiation, has the activities of promoting wound tissue repair, collagen regeneration and hair follicle regeneration, and can be applied to industries such as medical devices, cosmetics or culture media.

[0034] In some alternative embodiments, the number of the basic repeating units is 3; The amino acid sequence of the recombinant humanized type XVII collagen (denoted as PSQ12x3) is shown in SEQ ID NO.2: GSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAK (SEQ ID NO.2).

[0035] In some alternative embodiments, an affinity tag is connected to the N-terminus of the recombinant humanized type XVII collagen.

[0036] An affinity tag is a technology that uses gene cloning means to fuse a polypeptide, protein domain or even a complete protein with specific functions with the target protein to achieve applications such as expression purification, detection and tracing of the target protein. Protein tags can be roughly classified into three categories according to their functions: detection tags, expression purification tags and tracing tags. Currently commonly used expression purification tags include His, GST, etc.

[0037] In a second aspect, the present invention provides a nucleic acid molecule that encodes the recombinant humanized type XVII collagen described above.

[0038] Nucleic acid molecules are the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). They are biological macromolecular compounds polymerized from many nucleotide monomers and are one of the most basic substances of life. The nucleotide sequence refers to the arrangement order of bases in DNA or RNA. The nucleic acid molecule contains cDNA. In some cases, the nucleic acid molecule can be modified for use in the vectors of the present 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 site sequences. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of nucleic acid-encoding sequences within or isolated from one or more given cells.

[0039] In a specific embodiment, the above nucleic acid molecule can optimize the relevant gene sequence according to the codon preference of the host cell and then synthesize the gene fragment artificially. It should be understood that nucleic acid molecules capable of translating into the above amino acid sequence are all within the protection scope of the present invention.

[0040] In some alternative embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.3: GGTTCTCCTGGTCCGCAGGGTCCACCTGGTCCGGTTGGTCTGCAGGGTCTGCGTGGTGAAGTGGGCCTGCCAGGTGTGAAAGGTGACAAAGGTCCGATGGGTCCGCCAGGTCCGAAAGGTGACCAGGGCGAGAAAGGTCCGCGTGGTCTGACCGGTGAACCTGGCATGCGCGGTCTGCCTGGTGCTGTGGGTGAACCAGGTGCAAAGGGCTCTCCAGGTCCACAGGGTCCGCCAGGTCCAGTAGGTCTGCAGGGTCTGCGCGGCGAGGTTGGTCTGCCTGGTGTAAAAGGTGACAAAGGTCCTATGGGTCCGCCGGGTCCGAAAGGTGATCAAGGTGAGAAGGGTCCGCGTGGTCTGACGGGCGAACCTGGTATGCGTGGCCTGCCAGGTGCAGTTGGTGAACCAGGCGCTAAAGGTTCTCCTGGTCCGCAAGGTCCACCAGGCCCTGTTGGTCTGCAGGGCCTGCGTGGTGAAGTTGGTCTGCCTGGCGTTAAAGGTGATAAAGGTCCGATGGGCCCACCGGGTCCTAAAGGTGATCAGGGTGAGAAAGGTCCACGTGGTCTGACTGGTGAACCGGGTATGCGTGGTCTGCCGGGTGCTGTTGGTGAACCAGGTGCAAAA (SEQ ID NO.3).

[0041] In a third aspect, the present invention provides a vector, and the vector carries the nucleic acid molecule described above.

[0042] In a fourth aspect, the present invention provides a cell, and the cell carries the nucleic acid molecule described above, or contains the vector described above, or expresses the recombinant humanized type XVII collagen.

[0043] In some alternative embodiments, the cell includes, but is not limited to, Escherichia coli, or other cells well known to those skilled in the art.

[0044] In a fifth aspect, the present invention provides a method for preparing recombinant humanized type XVII collagen, including: expressing the recombinant humanized type XVII collagen by using the cell described above, and then preparing the recombinant humanized type XVII collagen through separation and purification.

[0045] Sixth aspect, the present invention provides the application of recombinant humanized type XVII collagen in medical devices or cosmetics.

[0046] Seventh aspect, the present invention provides the application of recombinant humanized type XVII collagen in culture media.

[0047] Through the research of the inventors, it is found that the recombinant humanized type XVII collagen provided by the present invention has good water solubility, high thermal stability, is not easily degraded, has good activities of cell adhesion, migration and proliferation, promotes the ability of vascular endothelial cells to form tubes, has no hemolytic reaction, has no sensitization after subcutaneous injection, has no risk of rejection, and has good biocompatibility; it has the effects of promoting the repair of skin barrier damage induced by ultraviolet radiation and inhibiting the increase of oxygen free radicals and inflammatory factors induced by ultraviolet radiation, and has the activities of promoting wound tissue repair, collagen regeneration and hair follicle regeneration, and can be applied to industries such as medical devices, cosmetics or culture media.

[0048] The present invention will be further illustrated by specific examples below. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0049] Example 1 This example introduces a preparation method of a recombinant humanized type XVII collagen fragment, taking PSQ12x3 as an example (the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.3). The specific steps are as follows: (1) Construction of recombinant expression plasmid According to the nucleotide sequence of SEQ ID NO.3, Nanjing GenScript Biotech Corporation was commissioned to synthesize the corresponding gene fragment. The synthesized gene fragment was inserted into the pET-32a(+) vector through the KpnⅠ and XhoⅠ restriction enzyme sites, thereby constructing a recombinant expression plasmid. The specific plasmid structure is as Figure 1 shown.

[0050] (2) Transformation of Escherichia coli competent cell BL21(DE3) The constructed recombinant expression plasmid was transformed into Escherichia coli BL21(DE3) competent cells. The transformation process includes: a. Add 100 ng of the recombinant plasmid to 100 μl of competent cells, mix gently and incubate on ice for 30 minutes; b. Perform heat shock treatment at 42 °C for 1 minute, and then cool the mixture on ice for 3 minutes; c. Add 900 μl of LB medium (containing 10 g / L sodium chloride, 10 g / L tryptone, 5 g / L yeast extract) to the cell mixture, and incubate with shaking at 37 °C and 200 rpm for 1 hour; d. Centrifuge the mixed solution at 8000 rpm for 3 minutes. After removing the supernatant, resuspend the cells and spread them on an LB plate containing sodium ampicillin. e. Invert the plate and incubate it at 37 °C for about 12 hours until monoclonal colonies grow.

[0051] (3)Induce the expression of recombinant protein Pick monoclonal colonies from the LB plate to induce the expression of recombinant humanized type XVII collagen fragment. The specific steps are as follows: a. Inoculate the monoclonal colonies into an LB medium containing sodium ampicillin and shake culture overnight at 37 °C. b. Inoculate the overnight culture solution into a fresh LB medium containing sodium ampicillin at an inoculation amount of 1% and continue to shake culture at 37 °C until the OD 600 value reaches 0.6 - 0.8. c. Add the inducer IPTG to a final concentration of 0.5 mM and shake culture at 16 °C for 16 - 24 hours to induce protein expression.

[0052] (4)Recovery, digestion and purification of recombinant protein Treat the bacterial solution obtained after induction to recover and purify the recombinant humanized collagen fragment. The specific steps include: a. Centrifuge the induced bacterial solution at 4000 rpm for 20 minutes at 4 °C, collect and wash the cell precipitate. b. Resuspend the cell precipitate with lysis buffer (20 mM imidazole, 500 mM sodium chloride, 20 mM PB, pH 7.4) and use a high-pressure homogenizer to break the cells. c. Centrifuge the broken bacterial solution at 12000 rpm for 60 minutes at 4 °C and collect the supernatant. d. The supernatant is subjected to affinity chromatography through a nickel column, and the impurity proteins are eluted with equilibration buffer (20 mM imidazole, 500 mM sodium chloride, 20 mM PB, pH 7.4). e. Elute the target protein with washing buffer (400 mM imidazole, 500 mM NaCl, 20 mM PB, pH 7.4). The specific purification situation is as Figure 2 shown. f. Exchange the buffer of the purified protein using a tangential flow ultrafiltration system, add an appropriate amount of TEV protease, and digest it overnight at 4 °C (see the digestion situation in Figure 3 ). g. The digested protein solution is subjected to affinity chromatography through a nickel column again to remove TEV protease and non-collagen parts. The specific impurity removal situation is shown in Figure 4 .

[0053] (5) Removal of endotoxin Finally, the endotoxin in the protein was removed by desalting buffer exchange and affinity chromatography. The specific steps are as follows: a. The purified protein solution was subjected to desalting buffer exchange using 20 mM Tris-base (pH 7.8); b. The protein sample was loaded onto a Q column, and the effluent was collected to remove endotoxin; c. The above steps were repeated until the endotoxin content in the protein met the standard (see the gel electrophoresis result of the sample after endotoxin removal in Figure 5 ).

[0054] Example 2 SDS-PAGE analysis of recombinant humanized type XVII collagen fragment In this example, the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example 1 was detected by SDS-PAGE analysis to determine its molecular weight range. The specific steps are as follows: (1) 15 μl was taken from the protein stock solutions prepared in each step of Example 1 (after precipitation and washing, a small amount of precipitate was taken and resuspended in the same volume of PBS), and 3 μl of 6× protein loading buffer (TransGen Biotech Co., Ltd.) was added. After the mixture was thoroughly mixed, it was heated in a water bath at 100 °C for 10 minutes.

[0055] (2) 10 μl of the loading solution and protein Marker (Xinsaimai Biotechnology Co., Ltd.) were respectively loaded into the SDS-PAGE gel wells, the voltage was set at 120 V, and electrophoresis was carried out for about 2 hours.

[0056] (3) After electrophoresis was completed, the protein gel was removed, rinsed three times with water, and then immersed in the protein staining solution (Beyotime Biotechnology Co., Ltd.) for 20 minutes. After staining, it was rinsed with water overnight, and the electrophoresis result is as shown in Figures 2 - 5 .

[0057] Example 3 In this example, mass spectrometry was used to detect the peptide coverage of the recombinant humanized type XVII collagen (PSQ12x3) produced in Example 1. This detection was completed by Beijing Bio-Tech Pack Technology Co., Ltd. The experimental method and experimental results are as follows: (1) Weighed 2 mg of collagen, added 200 μL of water, and dissolved it thoroughly. The collagen was enriched by SP3 magnetic beads.

[0058] (2)The dried magnetic beads were separately digested with Trypsin and Glu-C at 37 °C with shaking for 16 hours. After digestion, the supernatant was taken and desalted using a self-packed desalting column, and the solvent was evaporated to dryness in a vacuum centrifuge concentrator at 45 °C. The peptide fragments were dissolved in a sample dissolution solution (0.1% formic acid, 2% acetonitrile), and after shaking and centrifugation, the supernatant was transferred to a sample loading tube and awaited mass spectrometry analysis.

[0059] (3)Mass spectrometry analysis was performed using an Easy-nLC1000 / Orbitrap Exploris 240 high-resolution liquid chromatography-mass spectrometry system for sample loading and data acquisition.

[0060] (4)Data analysis was carried out using Thermo BioPharma Finder software, and the search parameters were as follows: the analysis database was the target protein sequence; trypsin digestion and serine protease digestion; variable modifications included protein N-terminal acetylation (Acetylation(Protein N-term)) and methionine oxidation (Oxidation (M)).

[0061] After database search and analysis, 100% of the sequences of the detected samples covered 100% of the target protein sequence, proving the successful expression of the recombinant protein PSQ12x3.

[0062] The results of the polypeptide coverage rate were as follows:

[0063] Example 4 In this example, the cell adhesion promoting activity of the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example 1 was detected. The specific steps were as follows: (1)Collagen solutions with target concentrations (0.1, 1, 10, 100, 1000 μg / ml) were prepared using PBS buffer; ensure that the collagen was completely dissolved in PBS to avoid precipitation.

[0064] (2)According to the experimental protocol, 100 μl of collagen solutions with different concentrations were added to a 96-well plate, and two columns were added with PBS without collagen as a control group. The 96-well plate was placed in an environment at 4 °C for overnight coating.

[0065] (3)The next day, the 96-well plate was taken out, the coating solution was removed, and 100 μl of PBS solution containing 5% BSA was added, and incubated at 37 °C for 1 hour for blocking.

[0066] (4)After blocking, the 96-well plate was taken out and washed once with PBS, and 20,000 HACAT cells were added to each well, with 90 μl of cell suspension (cell suspension diluted with serum-free medium) in each well.

[0067] (5) According to the cell adhesion situation, after 20 - 40 min, take out the 96 - well plate. Leave one group as the control group without any treatment. For the other control group and the remaining experimental groups, aspirate the supernatant and wash once with PBS.

[0068] (6) Add 100 μl of serum - free medium containing CCK8 (medium:CCK8 = 9:1). For the control group without treatment, directly add 10 μl of CCK8.

[0069] The results are as Figure 6 shown. Based on the average value and standard error of 3 parallel experiments, plot the graph.

[0070] Figure 6 The results show that compared with the cell adhesion rate of the control group PBS (26.0% ± 3.56%), when the concentrations of the recombinant humanized type XVII collagen PSQ12x3 prepared in Example 1 are 1 μg / mL, 10 μg / mL, 100 μg / mL, and 1000 μg / mL, the cell adhesion rates are 45.33% ± 6.02%, 62.67% ± 4.19%, 67.00% ± 6.53%, and 65.00% ± 2.94% respectively. Compared with the PBS group, they are increased by 74.36% ( P < 0.01), 141.03% ( P < 0.01), 157.59% ( P < 0.01), and 150.00% ( P < 0.01), indicating that PSQ12x3 has good cell adhesion characteristics.

[0071] Example Five In this example, the cell proliferation rate of the recombinant humanized type XVII collagen (PSQ12x3) prepared in Example 1 is detected. The specific steps are as follows: (1) Culture HACAT cells to about 90% confluence.

[0072] (2) Digest the cells with trypsin. After taking an appropriate amount of cell suspension, inoculate it into a 96 - well plate at a density of 5000 cells per well and allow it to adhere overnight.

[0073] (3) The next day, according to the experimental design, prepare media containing different concentrations (0.1, 0.5, 1, 5 mg / mL) of collagen, and use human type III collagen (Advanced BioMatrix, derived from human embryos) as the positive control group.

[0074] (4) Take out the 96 - well plate, replace it with the newly prepared medium, and continue culturing.

[0075] (5) After 24 - hour culture, use the CCK8 kit to detect cell proliferation.

[0076] The results are as Figure 7 shown, and the graph is plotted based on the average value and standard error of 3 parallel experiments.

[0077] Figure 7 The results show that the recombinant humanized type XVII collagen PSQ12x3 prepared in Example 1 can promote the proliferation of HACAT cells at 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, and 5 mg / mL, and has no obvious effect on cell viability. Human type III collagen shows a significant concentration-dependent cell proliferation inhibitory effect.

[0078] Example 6 In this example, the cell migration-promoting effect of the recombinant humanized type XVII collagen (PSQ12x3) prepared in Example 1 was detected. The steps are as follows: (1) Prepare a collagen solution with a target concentration (50 μg / mL) using sterile PBS.

[0079] (2) According to the experimental protocol, add 1 mL (50 μg / mL) of the collagen solution to a 12-well plate and place it at 4 °C overnight for coating.

[0080] (3) The next day, take out the 12-well plate, aspirate the supernatant, add PBS containing 5% BSA, and incubate at 37 °C for 1 h for blocking.

[0081] (4) After blocking, wash twice with PBS, and then dry the plate at 37 °C.

[0082] (5) After drying, place an ibidi scratch insert (four chambers) in each well, inoculate 2×10 4 cells in each chamber, and place it back in the 37 °C incubator to incubate overnight to allow the cells to adhere.

[0083] (6) Take out the 12-well plate on the second day, carefully remove the insert, replace the medium containing 1% FBS, and record the initial scratch image.

[0084] (7) After continuing to incubate overnight at 37 °C, take a photo again. As shown in Figure 8 a, use ImageJ software to analyze and calculate the area of the cell migration region, calculate the relative healing rate, and the results are as shown in Figure 8 b.

[0085] The results are as Figure 8 shown, and the graph is plotted based on the average value and standard error of 3 parallel experiments.

[0086] Figure 8The results showed that, compared with the control group, 50 μg / ml collagen significantly promoted the migration of HACAT cells, and the relative healing rate increased by about 87.62% compared with the control group ( P < 0.001).

[0087] Example VII In this example, a hemolysis experiment was performed on the recombinant humanized type XVII collagen (PSQ12x3) prepared in Example 1 to evaluate its safety. The specific method is as follows: (1) Collect an appropriate amount of fresh anticoagulated mouse whole blood, centrifuge at 3000 rpm for 10 min, and discard the supernatant. Wash the red blood cells twice with physiological saline (3000 rpm, 5 min each time), and finally resuspend the red blood cells in physiological saline to prepare a 2% (v / v) red blood cell suspension.

[0088] (2) The experiment was grouped according to different concentrations of the PSQ12x3 solution. Add 50 μL of 2% red blood cell suspension to 950 μL of the PSQ12x3 solution (final concentrations of 10, 100, 1000 μg / mL), and gently mix. In addition, a negative control group (replacing PSQ12x3 with physiological saline) and a positive control group (replacing PSQ12x3 with 0.1% Triton X-100) were set up for reference in hemolytic evaluation.

[0089] (3) Incubate all samples in a 37°C constant temperature water bath for 1 h. After incubation, centrifuge at 3000 rpm for 5 min, take 100 μL of the supernatant and transfer it to a 96-well plate. Measure the absorbance (OD value) of hemoglobin released by hemolysis at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The negative control group (physiological saline) was defined as 0% hemolysis, and the positive control group (0.1% Triton X-100) was defined as 100% hemolysis, and the hemolysis rate was calculated.

[0090] The results are as Figure 9 shown, and the mean and standard error of 3 parallel experiments were plotted.

[0091] As Figure 9 shown, PSQ12x3 did not cause significant hemolytic reactions at the tested concentrations of 10, 100, 1000 μg / mL. Compared with the negative control group (physiological saline), there was no significant difference in the hemolysis rate of each concentration of the PSQ12x3 group, and it was significantly lower than that of the positive control group (Triton X-100) ( P < 0.001). The above results indicate that within the tested concentration range, PSQ12x3 does not induce hemolysis of mouse red blood cells, further demonstrating its good biocompatibility and blood safety.

[0092] Example VIII In this example, an in vivo sensitization experiment was conducted on the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example 1, and the method for evaluating its sensitization by detecting the IgE level around the skin at the injection site was as follows: (1) The mice were randomly divided into three groups: the experimental group (PSQ12x3 solution, PSQ12x3 solution + aluminum adjuvant), the positive control group (ovalbumin + aluminum adjuvant), and the negative control group (saline + aluminum adjuvant).

[0093] (2) On day 0, after disinfecting the back skin of the mice with an alcohol cotton ball, 100 μL of the corresponding reagent was locally injected subcutaneously: the experimental group was injected with PSQ12x3 solution (1000 μg / mL) or PSQ12x3 solution + aluminum adjuvant respectively, the positive control group was injected with ovalbumin solution (100 μg / mL + aluminum adjuvant), and the negative control group was injected with saline + aluminum adjuvant. All reagents were pre-mixed with aluminum adjuvant (except for the PSQ12x3 alone group). The injection sites were set on both sides of the midline of the mice's back, alternating left and right to reduce local stress responses. The same immunization injection was repeated on day 14 to enhance the immune response.

[0094] (3) After injection, the local reactions such as erythema and induration at the injection site on the back of the mice were observed and recorded daily. On day 3, mice in the group injected with PSQ12x3 solution alone were randomly selected, and skin tissue samples were taken from the injection site for HE staining to observe the infiltration of inflammatory cells and evaluate the local immune response.

[0095] (4) On day 28, after anesthetizing the mice, skin tissue samples were taken from around the injection site to ensure that the epidermis and dermis were included. The tissue samples were placed in pre-cooled PBS, homogenized to extract tissue fluid, and used for subsequent enzyme-linked immunosorbent assay (ELISA) to detect the expression levels of IL6, IL4, TNFα, and IL17A in the skin tissue. Blood was collected synchronously, serum was separated, and ELISA was used to detect the specific IgE level in the serum of each group of mice. The experiment was strictly operated according to the kit instructions. Finally, the absorbance (OD value) was measured by a spectrophotometer, and the concentrations of IL6, IL4, TNFα, IL17A in the skin tissue and serum IgE of each group were calculated and compared to evaluate the immunogenicity and sensitization of PSQ12x3.

[0096] The results are as Figure 10 shown, and the graph was plotted based on the average value and standard error of 3 parallel experiments.

[0097] The experimental results showed that after subcutaneous injection of PSQ12x3 solution (1000 μg / mL), no typical allergic reactions such as erythema and nodules appeared on the skin of the mice. Further HE staining analysis confirmed that the tissue structure at the injection site remained intact, and no infiltration of inflammatory cells or other histological abnormalities were observed ( Figure 10in a). ELISA test results showed that PSQ12x3 (1000 μg / mL) did not cause significant increases in serum IgE and the levels of IL-4, IL-6, TNFα, and IL-17A in tissues ( P > 0.05) ( Figure 10 in b- Figure 10 in f). These results indicated that PSQ12x3 had good biocompatibility and did not induce significant immune inflammation or allergic reactions, suggesting a low risk of sensitization.

[0098] Example Nine In this example, the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example One was subjected to an endothelial cell (HUVEC cell) tube formation assay to evaluate its safety. The specific method was as follows: (1) Dissolve PSQ12x3 at 10, 100, and 1000 μg / mL in endothelial cell medium (ECM), and place it in an incubator at 37 °C for 12 h to ensure that PSQ12x3 is fully dissolved and maintains stability.

[0099] (2) Take out Matrigel from the -20 °C refrigerator, slowly melt it overnight in a 4 °C refrigerator, and keep it on ice throughout the experimental operation to prevent premature solidification from affecting the experimental results.

[0100] (3) Use a pre-cooled pipette to evenly spread 50 μL of Matrigel into each well of a 96-well plate, and place it in an incubator at 37 °C for 30 min to allow Matrigel to completely solidify and form a stable matrix layer. The above experiments were all carried out on ice.

[0101] (4) Resuspend HUVEC cells in the logarithmic growth phase with ECM medium containing different concentrations of PSQ12x3, set the cell density to 2×10 5 cells / mL, and add 100 μL of the cell suspension (i.e., 2×10 4 cells per well) to each Matrigel-coated well. Set up a control group (using only ECM medium), and set 3 replicate wells for each group to ensure the repeatability and statistical reliability of the experiment.

[0102] (5) Place the 96-well plate seeded with cells in an incubator at 37 °C and 5% CO2 for 12 h to promote the attachment of HUVEC cells and the formation of tubular structures.

[0103] (6) After incubating for 12 h, use an inverted microscope to observe the tube formation ability of HUVEC cells in each experimental group ( Figure 11in a). Quantitatively analyze the number of tube connections, compare the tube formation ability between the PSQ12x3 treatment group and the control group to evaluate the biocompatibility and potential biological effects of PSQ12x3.

[0104] The results are as Figure 11 shown, and the graphs are plotted based on the mean and standard error of 3 parallel experiments.

[0105] The experimental results show that at all tested concentrations (10, 100, 1000 μg / mL), PSQ12x3 did not significantly affect the tube formation ability of HUVEC cells ( Figure 11 in a). Compared with the control group (only containing endothelial cell medium), each concentration of the PSQ12x3 treatment group could maintain the formation of normal microtubule structures. Microscopic observation and image analysis further confirmed that there was no statistical difference in the number of tube connections between the different concentration PSQ12x3 treatment groups and the control group ( P >0.05) ( Figure 11 in b). These results indicate that under the experimental conditions, PSQ12x3 has no obvious inhibitory effect on the angiogenesis ability of HUVEC cells, does not cause cytotoxicity or functional damage, and shows good biocompatibility.

[0106] Example Ten In this example, an experiment on the repair of ultraviolet (UV)-radiation-induced skin barrier damage was carried out on the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example One to evaluate the repair ability of PSQ12x3 on damaged skin and its effect on the expression of skin barrier-related proteins. The specific method is as follows: (1) The experimental mice were randomly divided into a normal control group (not receiving UV irradiation, only applying PBS); a UV damage group (i.e., the model group, receiving UV irradiation, without PSQ12x3 intervention, only applying PBS), and a PSQ12x3 treatment group (after UV irradiation, applying 1 mg / mL PSQ12x3 every day) for 7 consecutive days.

[0107] (2) UVA (10 J / cm 2 ) + UVB (100 mJ / cm 2 ) were used to irradiate the back skin of the mice once a day to induce skin barrier damage. The mice in the normal control group received non-UV irradiation (light source off) for the same time. After the UV irradiation was completed, 100 μL of PSQ12x3 (1 mg / mL) was evenly applied to the damaged area of the mice in the PSQ12x3 treatment group for 7 consecutive days to evaluate the promoting effect of PSQ12x3 on skin barrier repair.

[0108] (3)Observe and record the skin damage conditions of the mice daily, including phenotypic characteristics such as erythema, dryness, and chapping, and take skin images using a high-resolution imaging system.

[0109] (4)Seven days after applying PSQ12x3, take the skin tissue on the back of the mice, and use two-photon excited fluorescence microscopy (TPEF) combined with second harmonic generation (SHG) technology to perform label-free real-time detection of the three-dimensional microstructure and collagen fiber distribution of the skin tissue. The experiment uses a Zeiss LSM 880 NLO microscope equipped with a Ti:Sapphire femtosecond laser (Chameleon Vision II, Coherent, with adjustable central wavelength). SHG imaging is used to analyze the arrangement, density, and morphology of collagen in the dermis. The excitation wavelength is set at 880 nm, and the SHG signal is collected using a 445 ± 20 nm bandpass filter. The grating spectral resolution is set at 8 nm. During image acquisition, a 40× water immersion objective lens (NA 1.2) is used, the Z-axis step size is set at 1 μm, and optical sections with a depth of 30 μm are collected, covering a 500 × 500 μm 2 field of view.

[0110] (5)Take the skin tissue on the back of the mice (5 × 5 mm 2 ) seven days after applying PSQ12x3, extract total RNA using Trizol, and measure the RNA concentration and purity (A260 / A280) using Nanodrop. After reverse transcription to synthesize cDNA, SYBRGreen fluorescence quantitative PCR is used to detect Flg, Cldn1 the mRNA expression level. Using GAPDH as an internal reference gene, the relative mRNA expression is calculated using the ΔΔCt method, and the gene expression differences between each experimental group are compared to evaluate the regulatory effect of PSQ12x3 on the transcriptional level of skin barrier protein genes.

[0111] The results are as Figure 12 shown, plotted based on the mean and standard error of 3 parallel experiments.

[0112] The experimental results show that in the ultraviolet-induced skin damage model, the skin of the mice in the model group showed significant erythema and roughness. After treatment with PSQ12x3 (1 mg / mL), the skin appearance was significantly improved compared to the model group, and the degree of erythema and damage was significantly reduced ( Figure 12 in a). The two-photon microscopy imaging results show that the collagen fiber structure in the dermis of the model group was disordered, and the SHG signal intensity was significantly weakened, indicating severe damage to collagen fibers. In contrast, the collagen fiber signal intensity in the PSQ12x3 treatment group was significantly enhanced, and the fiber arrangement tended to be uniform, indicating that PSQ12x3 has a significant effect on collagen fiber repair ( Figure 12in b). The qPCR test results showed that the mRNA expression levels of the skin barrier function-related genes Claudin - 1 and Filaggrin in the PSQ12x3 treatment group were increased by 43.78% ( P <0.01) and 35.38% ( P <0.01) respectively compared with the model group ( Figure 12 in c). These results indicated that PSQ12x3 could effectively improve the ultraviolet-induced skin damage, promote the reconstruction of collagen fibers, and enhance the expression of skin barrier-related genes, showing significant potential for skin repair.

[0113] Example XI In this example, an experimental study on the ultraviolet (UV) radiation-induced oxidative stress model of the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example I was carried out to evaluate the inhibitory effect of PSQ12x3 on the level of oxygen free radicals in damaged skin and its potential antioxidant protection ability. The specific experimental methods are as follows: (1) The experimental mice were randomly divided into a normal control group (not receiving UV irradiation, only applying PBS); a UV damage group (receiving UV irradiation, without PSQ12x3 intervention, only applying PBS) and a PSQ12x3 treatment group (after UV irradiation, applying 1 mg / mL PSQ12x3 every day), for 7 consecutive days.

[0114] (2) UVA (10 J / cm 2 ) + UVB (100 mJ / cm 2 ) were used to irradiate the back skin of the mice once a day to induce skin barrier damage. The mice in the normal control group received non-UV irradiation (light source off) for the same time. After the UV irradiation was completed, 100 μL of PSQ12x3 (1 mg / mL) was evenly applied to the damaged area of the mice in the PSQ12x3 treatment group for 7 consecutive days to evaluate the inhibitory effect of PSQ12x3 on the level of oxygen free radicals in damaged skin and its potential antioxidant protection ability.

[0115] (3) After the application of PSQ12x3 was completed (on the 7th day), the mice were deeply anesthetized and the back skin tissues were immediately collected. The surface of the samples was gently washed with pre-cooled PBS to remove excess liquid and impurities. The washed tissues were placed in pre-cooled OCT embedding medium and quickly frozen at -80 °C. Subsequently, continuous tissue sections with a thickness of 8 μm were prepared using a cryostat. The prepared sections were stored in a -80 °C refrigerator for subsequent experiments.

[0116] (4)Detect the level of oxygen free radicals in tissues using the DCFH-DA fluorescent probe. During the experiment, the frozen sections were equilibrated to room temperature and gently washed with PBS buffer to remove the residual OCT embedding medium. Subsequently, the surface of the sections was covered with a 10 μM DCFH-DA working solution (diluted with serum-free medium) to ensure full coverage of the staining area. The sections were incubated at 37 °C in the dark for 30 minutes to promote the reaction of the probe with intracellular reactive oxygen species. After incubation, the sections were gently washed 3 times with PBS buffer for 5 minutes each to remove the unbound free dye and minimize background fluorescence interference.

[0117] (5)Detect and record the DCFH-DA staining results using a fluorescence microscope ( Figure 13 a in). The excitation wavelength was set to 488 nm, the emission wavelength was set to 525 nm, a 20× objective lens was selected to magnify the field of view, and fluorescence images of at least 3 sections were randomly collected. Image quantitative analysis was performed using ImageJ software ( Figure 13 b in), and the mean fluorescence intensity (MeanFluorescence Intensity) of each field of view was calculated as an index of the level of oxygen free radicals.

[0118] The results are shown as Figure 13 a graph of the mean value and standard error based on 3 parallel experiments.

[0119] The experimental results showed that the DCFH-DA fluorescence signal in the skin tissue of the model group (UV radiation group) was significantly enhanced, reflecting a significant increase in the level of oxygen free radicals induced by UV radiation; after treatment with PSQ12x3 (1 mg / mL), the fluorescence signal intensity of the skin tissue was significantly lower than that of the model group and close to the control group level, indicating that PSQ12x3 has the potential to inhibit oxidative stress induced by UV radiation in skin tissue. Further quantitative analysis results showed that the mean fluorescence intensity of the PSQ12x3 treatment group was reduced by approximately 43.60% compared with the model group ( P <0.01), suggesting that PSQ12x3 can effectively alleviate the increase in oxygen free radicals caused by UV radiation and play a significant antioxidant protection role.

[0120] Example XII In this example, an experiment on HUVEC damage induced by ultraviolet (UV) radiation was carried out on the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example 1 to evaluate the effects of PSQ12x3 on the generation of oxygen free radicals, the secretion of inflammatory factors, and the angiogenesis ability of damaged HUVEC. The specific experimental methods are as follows: (1) Select HUVEC cells in the logarithmic growth phase and culture them according to routine aseptic procedures. Use endothelial cell medium (ECM) containing 10% fetal bovine serum (FBS) and 1% double antibody (penicillin - streptomycin). When the cell state is good, gently digest the cells with 0.25% trypsin - EDTA, and after centrifugation, adjust the cell density to 2×10 5 cells / mL.

[0121] (2) Randomly divide the HUVEC cells into a UV - damaged group (cultured in a conventional medium after UV radiation); a collagen III treatment group (cultured in a medium containing 100 μg / mL collagen III after UV radiation); a PSQ12x3 treatment group (cultured in a medium containing 100 μg / mL PSQ12x3 after UV radiation). Set 3 replicate wells for each group to ensure the reliability and statistical significance of the experimental data.

[0122] (3) Use a UV radiation device to perform UV treatment on the cells. Evenly spread the cells in the collagen III treatment group and the PSQ12x3 treatment group in culture dishes. After removing the culture dish lids, expose them to UVA (10 J / cm 2 ) and UVB (100 mJ / cm 2 ) radiation for 10 minutes to induce oxidative stress and inflammatory responses. The UV - damaged group uses the same radiation conditions. The cells in the normal control group receive sham irradiation (light source off) during the same time period to exclude the influence of other environmental interference factors.

[0123] (4) After the UV radiation treatment is completed, immediately gently wash the cells in each group twice with PBS buffer to remove residual UV - induced products and the culture medium. Subsequently, resuspend the cells in the corresponding culture medium, and add collagen III and PSQ12x3 with a final concentration of 100 μg / mL to the culture media of the collagen III treatment group and the PSQ12x3 treatment group respectively. The cells are cultured for another 12 hours for subsequent functional detection and molecular analysis.

[0124] (5) Evenly spread 50 μL of Matrigel in a 96 - well plate and incubate it in a 37°C incubator for 30 minutes to allow it to completely gel. Resuspend the treated HUVEC cells in the corresponding culture medium, adjust the cell density to 2×10 5 cells / mL, and add 100 μL of the cell suspension to each well. Incubate the 96 - well plate in a 37°C, 5% CO2 incubator for 12 hours to promote cell attachment and tubular structure formation.

[0125] After the incubation, an inverted microscope was used to observe the tube formation ability of HUVEC cells in each group, and images were taken in random fields of view. The number of nodes, branches, and total tube length of tube connections were recorded and statistically analyzed. Quantitative analysis of the images was performed using ImageJ software to evaluate the effects of collagen III and PSQ12x3 on the angiogenesis ability of HUVECs.

[0126] (7)The level of oxygen free radicals was detected using a DCF-DA fluorescent probe. After the culture ended, the cells in each group were washed twice with PBS, and then 10 μM DCF-DA working solution (diluted with serum-free medium) was added. The cells were incubated for 30 minutes at 37°C in the dark to ensure that the probe fully reacted with the intracellular reactive oxygen species. After the incubation, the cells were washed three times with PBS again to remove the unbound probe. Fluorescent images were observed and taken using a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm), and the fluorescence intensity was quantitatively measured using a fluorescence microplate reader. The results were expressed as the average fluorescence intensity of each group to represent the level of oxygen free radicals.

[0127] (8)After culturing for 12 hours, the culture supernatants and cell lysates of each group were collected. The supernatants were used to detect the secretion levels of inflammatory factors (IL-6 and TNF-α), and a commercial ELISA kit was used, operating strictly according to the kit instructions. Finally, the absorbance was read at a wavelength of 450 nm using a microplate reader, and the concentrations of inflammatory factors in each group were calculated.

[0128] The results are as Figure 14 shown, and plots were made based on the means and standard errors of 3 parallel experiments.

[0129] The experimental results showed that the tube formation ability of HUVEC cells in the PSQ12x3 treatment group was significantly better than that in the collagen III treatment group, and significantly inhibited the generation of oxygen free radicals in damaged HUVEC cells ( Figure 14 in a). The results of quantitative analysis showed that, as shown in Figure 14 b, the number of tube connections in the PSQ12x3 treatment group increased by approximately 755.56% compared with the collagen III treatment group ( P <0.001), and the average fluorescence intensity in the PSQ12x3 treatment group decreased by approximately 94.34% compared with the collagen III treatment group ( P <0.001). The ELISA test results showed that the concentrations of IL-6 and TNF-α in the PSQ12x3 treatment group were significantly lower than those in the collagen III treatment group ( P <0.05), indicating that PSQ12x3 has an inhibitory effect on the inflammatory response induced by UV radiation ( Figure 14 in d and Figure 14 in e).

[0130] Example Thirteen This example verifies the effect of the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example 1 on mouse skin wound repair. The specific experimental method is as follows: (1) The experimental mice were randomly divided into an experimental group (1 mg / mL PSQ12x3 group) and a negative control group (PBS group).

[0131] (2) The mice were anesthetized with anesthetic (2% isoflurane). A circular full-thickness skin wound with a diameter of about 1 cm was made on the back of each mouse using sterile surgical tools to ensure complete destruction of the skin barrier while avoiding damage to large subcutaneous blood vessels. After surgery, a sterile cotton swab was used to clean the wound area to prevent infection. 100 μL of the experimental solution was applied to the wound area: the experimental group was applied with 1 mg / mL PSQ12x3 solution, and the negative control group was injected with an equal amount of PBS. After application, the mice were housed individually to prevent licking and biting each other from interfering with wound healing. At the same time, the environment was kept clean and the bedding was changed regularly.

[0132] (3) On the 0th, 3rd, 6th, 9th and 12th day of the experiment, a high-definition digital camera was used to vertically record the wound healing process. Before shooting, the wound surface was gently cleaned with saline, and the diameter and area of the wound were measured with a vernier caliper. The wound area was calculated by analysis software, the wound healing speed was quantified, and a curve of the wound area changing over time was drawn, such as Figure 15 As shown in a in .

[0133] (4) At the end of the experiment on the 12th day, the mice were painlessly killed after deep anesthesia, and wound and surrounding skin tissue samples were collected. After the samples were gently washed with PBS, they were immediately fixed in a 10% neutral formalin solution for 24 hours. Subsequently, gradient dehydration, clearing, and paraffin embedding were performed to prepare continuous tissue sections with a thickness of approximately 5 μm. HE staining and Masson staining were performed to evaluate epithelial regeneration and collagen deposition, such as Figure 15 b in.

[0134] The results are as follows Figure 15 As shown, the mean values and standard errors based on 3 parallel experiments are plotted.

[0135] The experimental results showed that the PSQ12x3 treatment group significantly promoted the wound healing of mice. On the 12th day, the wound healing rate reached 87.70%±4.71%, which was about 60.80% higher than that of the control group (54.54%±3.44%). P(<0.001). The HE staining results showed that the PSQ12x3 treatment group had a relatively complete epidermal structure and regular arrangement of epidermal cells. The collagen fibers in the dermis were densely distributed, the degree of inflammatory cell infiltration was significantly lower than that of the control group, the number of hair follicles increased significantly, the structure was more complete, and the surrounding matrix was well reconstructed, indicating more mature tissue repair and significantly reduced inflammatory response. The Masson staining results showed that the PSQ12x3 treatment group showed a large number of densely arranged collagen fibers, distributed in a wavy pattern, and the fiber arrangement direction was more regular, approaching that of normal skin tissue. In summary, PSQ12x3 significantly promoted epidermal regeneration and dermal tissue reconstruction at the wound site, manifested as reduced inflammatory response, proliferation of collagen fibers, and orderly arrangement.

[0136] Example XIV This example studied the effect of the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example 1 on promoting the proliferation of human hair follicle dermal papilla cells (HFDPC). The specific experimental methods are as follows: (1) Human hair follicle dermal papilla cells (HFDPC) were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibody (penicillin-streptomycin), and cultured in an environment of 37 °C and 5% CO2 saturated humidity. When the cell state was good, the cells were digested with 0.25% trypsin-EDTA, and the cell density was adjusted to 5×10 4 cells / mL.

[0137] (2) According to the experimental design, media containing different concentrations (10, 100, 1000 μg / mL) of collagen were prepared, and the control group was a normal medium.

[0138] (3) Cells were seeded in a 96-well plate at a density of 1×10 4 cells / well, and 100 μL of the corresponding medium was added to each well. The plate was cultured in an incubator at 37 °C and 5% CO2 for 24 hours to ensure cell attachment.

[0139] (4)After adherent growth, aspirate the culture medium, add the culture medium corresponding to the treatment group, and continue culturing for 48 hours. Replace the fresh culture medium once during the culture period to maintain stable experimental conditions. After the culture is completed, add the EdU working solution with a final concentration of 10 μM to the culture medium and continue culturing at 37 °C for 2 hours to ensure sufficient incorporation of EdU into the cell DNA. Subsequently, aspirate the culture medium, gently wash the cells 3 times with PBS for 5 minutes each time, fix them in 4% paraformaldehyde solution at room temperature for 20 minutes, and then wash 3 times with PBS. After fixation, add 0.5% Triton X-100 and incubate at room temperature for 10 minutes to increase the cell membrane permeability, and wash 3 times with PBS again. Conduct the experiment according to the instructions of the EdU color development kit, incubate at room temperature in the dark for 30 minutes to complete color development, and then wash 3 times with distilled water.

[0140] (5)Use an optical microscope to observe the nuclear staining ( Figure 16 a in). Under randomly selected high-power fields of view, take images of at least 3 fields of view and record the number of EdU-positive cells in each group.

[0141] The results are as Figure 16 shown. Plot the average value and standard error based on 3 parallel experiments.

[0142] The results show that compared with the control group, the PSQ12x3 treatment group significantly increased the cell proliferation rate at concentrations of 10, 100, and 1000 μg / mL respectively ( Figure 16 b in), with the increase amplitudes being 15.23% ± 4.00% ( P < 0.01), 30.70% ± 5.08% ( P < 0.01) and 21.17% ± 5.87% ( P < 0.01). Among them, PSQ12x3 at a concentration of 100 μg / mL showed the most significant promoting effect, and the proliferation rate reached the highest level.

[0143] Example XV This example studies the effect of the recombinant humanized type XVII collagen fragment (PSQ12x3) prepared in Example 1 on promoting hair follicle regeneration in mouse skin wound tissue. The specific experimental method is as follows: (1)Randomly divide the experimental mice into a normal control group (only apply PBS without undergoing wound modeling); a wound model group (establish a skin wound model but do not intervene with PSQ12x3, only apply PBS); a PSQ12x3 treatment group (on the basis of the skin wound model, apply 100 μg / mL PSQ12x3 solution daily for 14 days).

[0144] (2) The day before the experiment, the hair on the back of the mouse was shaved and the residual hair was removed with a depilatory cream to ensure the smoothness and uniformity of the experimental area. The mouse was anesthetized with an anesthetic (2% isoflurane) under sterile conditions. A circular full-thickness skin wound with a diameter of about 10 mm was made on the back of the mouse using sterile surgical tools, ensuring that the wound penetrated the epidermis and dermis, exposing the subcutaneous tissue while avoiding damage to the large blood vessels and muscle layer. After the wound was made, the wound was cleaned with sterile PBS to remove tissue debris and prevent contamination and secondary infection.

[0145] (3) After the wound model was established, the normal control group did not receive any treatment, the wound model group was treated with 100 μL PBS evenly on the wound surface, and the PSQ12x3 treatment group was treated with 100 μL 100 μg / mL PSQ12x3 solution evenly on the wound surface. Wound treatment was performed once a day for 14 days, and the general condition, body weight changes, and eating habits of the mice were observed.

[0146] (4) At the end of the experiment on the 14th day, the mice were deeply anesthetized and then painlessly killed. The wounds and surrounding skin tissues were removed and immediately fixed in 4% neutral formalin solution for 24 hours. After fixation, the tissues were dehydrated with gradient ethanol, cleared with xylene, and embedded in paraffin to prepare continuous tissue sections with a thickness of approximately 5 μm.

[0147] (5) Immunofluorescence staining analysis: Keratin 15 (K15, hair follicle stem cell marker) and β-catenin (β-catenin, a key protein in the hair follicle regeneration signaling pathway) were used to detect the expression of hair follicle regeneration-related proteins. After dewaxing, the paraffin sections were subjected to citric acid antigen retrieval, washed with PBS, and blocked with 5% BSA at room temperature for 1 hour to reduce nonspecific binding. Then, primary antibodies (Keratin 15 1:200, β-catenin 1:200) were added respectively, and incubated in a wet box at 4°C overnight. The next day, the sections were washed with PBS three times for 5 minutes each time, and then fluorescently labeled secondary antibodies (1:500) were added and incubated at room temperature for 1 hour in the dark.

[0148] (6) Cell nuclei were counterstained with DAPI, and the sections were washed with PBS and sealed. The hair follicle regeneration of each group was observed using a confocal microscope. Three high-power fields were randomly selected to take images under a high-power microscope, and the expression levels of keratin 15 and β-catenin fluorescence signals were recorded respectively.

[0149] The results are as follows Figure 17 As shown in the results, the expression of keratin 15 and β-catenin in the hair follicle area was obvious in the normal mouse group, while the expression of the control group was weakened and the distribution was disordered. The PSQ12×3 group significantly restored and enhanced the expression of keratin 15 and β-catenin, suggesting that it may have a potential repair effect on the structure or function of hair follicles.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A recombinant humanized type XVII collagen, characterized in that, The recombinant humanized type XVII collagen comprises a basic repeating unit, and the amino acid sequence of the basic repeating unit is as shown in SEQ ID NO.1; The number of the basic repeating units is at least 1. When the number of the basic repeating units is greater than or equal to 2, adjacent basic repeating units are connected by peptide bonds.

2. The recombinant humanized type XVII collagen according to claim 1, wherein The number of the basic repeating units is 3; the amino acid sequence of the recombinant humanized type XVII collagen is as shown in SEQ ID NO.

2.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant humanized type XVII collagen according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, wherein The nucleotide sequence of the nucleic acid molecule is as shown in SEQID NO.

3.

5. A carrier, characterized in that, The vector carries the nucleic acid molecule according to claim 3 or 4.

6. A cell, characterized in that, The cell carries the nucleic acid molecule according to claim 3 or 4, or contains the vector according to claim 5, or expresses the recombinant humanized type XVII collagen according to claim 1 or 2.

7. The cell according to claim 6, wherein The cell includes Escherichia coli.

8. A method for preparing recombinant humanized type XVII collagen, characterized in that, Comprising: Expressing the recombinant humanized type XVII collagen by using the cell according to claim 6 or 7, and then preparing the recombinant humanized type XVII collagen after separation and purification.

9. Use of the recombinant humanized type XVII collagen according to claim 1 or 2 in medical devices or cosmetics.

10. Use of the recombinant humanized type XVII collagen according to claim 1 or 2 in culture media.

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