A recombinant human type XVII collagen, its preparation method and application

Abstract

The present invention relates to a recombinant human type XVII collagen, its preparation method and application. The recombinant human type XVII collagen is any one of COL17A 3a, COL17A 3b, COL17A 3c, COL17A 3d or COL17A 3e. The present invention uses computer software to analyze the functional fragments of type XVII collagen, screen the minimum functional region of type XVII collagen, and uses an Escherichia coli recombinant expression system for expression and extraction, so as to obtain a target functional fragment with a high yield.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant human type XVII collagen, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen is the earliest discovered and most abundant extracellular matrix protein and is a key structural component of the extracellular matrix. It is widely present in tissues and organs such as skin, bone, tendon, teeth, internal organs (such as stomach, intestine, heart, lung, blood vessel and esophagus), ligament, sclera, and eye, accounting for 30%-40% of the human body content. The biological function of collagen is largely determined by its spatial structure. Different types of collagen have different physiological functions and structures. Only proteins with a structure above the tertiary level have physiological functions, while collagen has a complete four-dimensional spatial structure. Collagen is a triple helix structure composed of three α polypeptide chains, and each α chain is a left-handed helical peptide segment composed of G-X-Y (X and Y represent any amino acid residues other than Gly, X is often Pro, and Y is often Hyp) repeat units. The most common sequence among them is the "glycine-proline-hydroxyproline" sequence, accounting for about 12% of the entire "G-X-Y" sequence, the "glycine-proline-Y" or "glycine-X-hydroxyproline" sequence accounts for about 44%, and other "glycine-X-Y" sequences constitute the remaining 44%. And the number of these repeating tripeptides "glycine-X-Y" is close to 1 / 3 of the entire sequence, playing a major role in maintaining the structure of collagen. Under the interaction of amino acid residues, the three α chains form a stable triple helix structure in a right-handed superhelix manner with the same axis as the center. Collagen plays an important role in organ support and body function protection, and it is also the main factor for the toughness of connective tissue. Connective tissue binds all the cells in the body and connects them into tissues and organs. Therefore, collagen is important for the human body.

[0003] Currently, 28 different types of collagen have been discovered. Collagens in different races and tissues have different chemical compositions or configurations. According to the function of collagen, it can be divided into fibrillar collagen and non-fibrillar collagen. In the human body, the most common collagens mainly have four forms: Type I collagen has the largest quantity in the human body and is mainly present in adult skin and bone tissues; type II collagen is mainly present in cartilage tissues; type III collagen is mainly present in the skin, blood vessels, intima, intestine, and stomach organs of infants; type IV collagen is mainly present in the basement membranes of various tissues and organs, placenta, and lens.

[0004] Collagen XVII, also known as type XVII collagen or COL17, is a transmembrane protein. It was first discovered by Diaz et al. in the 1980s in Bullous Pemphigoid (BP). It is a type II transmembrane protein. Its most obvious structural feature is that the N-terminus is located inside the cell and consists of 501 amino acids, including 4 tandem repeat sequences composed of 24 - 26 amino acids. The C-terminus is a longer extracellular peptide bond that connects to the anchoring fibrils of the clear plate of the skin basement membrane. The extracellular collagen domain of Collagen XVII contains repeated "glycine-X-Y" triplets, where X is usually proline and Y is hydroxyproline. The intervening non-collagen domains are called NC1, NC2, up to NC16 starting from the carboxyl terminus (C-terminus). The higher-order structure of Collagen XVII is a trimer, driven by the assembly of the triple helix of the collagen domain. Immunoelectron microscopy studies show that in the basement membrane, the extracellular domain (ECD) of Collagen XVII passes through the lamina lucida of the basement membrane and enters the lamina densa, and then bends back to the lamina lucida. Rotary shadow electron microscopy imaging also proves that its extracellular domain (ECD) has a rod-like morphology, while the intracellular domain part (ICD) with basic properties is spherical.

[0005] Collagen type XVII is a structural component of hemidesmosomes and is abundantly expressed in skin epithelial cells and other stratified epithelial cells, such as oral mucosa, cornea, cervix, and vagina. It is also present in large amounts in the squamous epithelial cells, syncytial cells, and cytotrophoblastic cells of the placenta. Collagen type XVII in the oral cavity is essential for enamel formation. Collagen type XVII is expressed at low levels in the mammary gland and intestinal epithelium. In layered epithelia, collagen type XVII is mainly expressed in basal cells. In the skin, it is present in both hair follicles and dermal-epidermal junctions. In addition to hemidesmosomal expression, non-hemidesmosomal collagen type XVII is also present on the apical surface of the soluble membrane of basal keratinocytes. Hemidesmosomes are the main junctional structures between basal cells and the underlying basement membrane zone, formed by the interdigitation of irregular protrusions of the dermal side of keratinocyte cell membranes with the basement membrane zone. Hemidesmosomes function to anchor cells to the basement membrane, thereby strengthening cell-cell connections. Hemidesmosomes decrease with age and lead to microseparation of basal cells. Among all hemidesmosomal components, collagen type XVII is the only one that significantly decreases during aging. Current studies also suggest that the instability of hemidesmosomes is caused by genomic instability due to intrinsic aging and UV-induced photoaging, leading to the hydrolysis of collagen type XVII. Exposure to UVB causes skin photoaging, resulting in pigmentation and hemidesmosome damage in both murine and human epidermis. Mice lacking collagen type XVII have more obvious skin pigmentation and slow wound healing; while overexpression of collagen type XVII can significantly reduce pigmentation and promote wound healing.

[0006] Collagen XVII plays an important role in maintaining the connection between intracellular and extracellular structural elements that maintain epidermal cell adhesion. The extracellular domain (ECD) of collagen XVII directly binds to integrin α6, laminin 332, and collagen IV. These interactions mediate the adhesion of keratinocytes to the basement matrix and regulate keratinocyte migration together with the shedding of the extracellular domain (ECD). In addition, studies have shown that collagen XVII may bind to or interact with periplakin, DFNB31, CSTF2T, PPLIL1, PLOD3, CDH1, PNKP, HRAS, SPDL1, EGFR, and ubiquitilin2. These interaction groups may be involved in the biosynthesis, transport, and post-translational modification of collagen XVII or its non-hemidesmosomal functions.

[0007] Studies have shown that skin aging is closely related to the self-renewal and functional differentiation of adult stem cells. The maintenance of the stem cell niche in the skin (such as epidermal stem cells, hair follicle stem cells, melanocyte stem cells) is crucial for the function of stem cells. The adhesion between cells and between cells and the extracellular matrix (ECM) is crucial for the establishment and maintenance of the stem cell niche structure. Adhesion to the underlying extracellular matrix is considered an important factor in maintaining epidermal stem cells (ESCs). Collagen XVII plays an important role in the maintenance of the stem cell niche. Therefore, collagen XVII plays an important role in skin regeneration and anti-aging.

[0008] Hair loss is one of the common manifestations of skin aging. Hair follicle stem cells (HFSCs) are responsible for the cyclic regeneration of hair follicles. Hair follicle stem cells receive signals from the surrounding environment and actively send signals to regulate the organization and function of their own niche. With the aging of hair follicle stem cells, the DNA damage response (DDR) is prolonged, which can activate ELANE (neutrophil elastase: an enzyme present in neutrophils, involved in immune responses and inflammatory processes, and capable of degrading various proteins), leading to the hydrolysis and loss of collagen COL17, a key molecule for maintaining HFSCs, thereby triggering the senescence of HFSCs. Aging hair follicle stem cells (HFSCs) terminally differentiate into epidermal keratinocytes through the epidermis and are periodically cleared from the skin. With the repetition of this atypical division, hair follicle stem cells (HFSCs) separate from the basement membrane, the hair follicle stem cell niche gradually shrinks, and the hair follicle itself becomes smaller (miniaturization), ultimately leading to hair thinning and hair loss. Studies have shown that the forced maintenance of collagen COL17 expression can inhibit the senescence of hair follicle stem cells, thereby delaying excessive hair loss.

[0009] At present, collagen is widely used in cosmetics, skin care, anti-aging and tissue repair. Currently, the collagen products sold on the market are mainly extracted from animal tissues such as pigs, cows, fish, etc. However, it is difficult to avoid the risks of virus infection and sensitization in the exogenous extraction of collagen. Moreover, the collagen extracted by conventional methods has strong coagulation function, which brings great risks in the application of tissue products. In addition, as a transmembrane protein, type XVII collagen is difficult to secrete and express, and the low expression level and difficulty in large-scale production also pose great challenges to the development and application of type XVII collagen. Therefore, the extraction and expression of recombinant collagen by genetic engineering technology is sustainable, which can greatly simplify the extraction process and reduce the extraction difficulty.

[0010] Although the efficacy of type XVII collagen in the fields of anti-aging and anti-hair loss has been confirmed, little is known about its main core functional fragments. In this invention, the minimum active functional fragment of type XVII collagen is found by using computer simulation and artificial intelligence methods, and then the active fragment is expressed by using a recombinant expression system, and it is found that it has excellent biological activity. Its production method is simple, fast, and easy to prepare on a large scale. It can be widely used in the treatment of skin anti-aging, anti-hair loss, gray hair and other fields. Summary of the Invention

[0011] Aiming at the problems existing in the prior art, the purpose of the design of this invention is to provide a recombinant human type XVII collagen and its preparation method and application, which are specifically realized through the following technical solutions:

[0012] In the first aspect of this invention, a recombinant human type XVII collagen is provided, and the recombinant human type XVII collagen is any one of COL17A3a, COL17A3b, COL17A3c, COL17A3d or COL17A3e.

[0013] Furthermore, the amino acid sequence of COL17A3a is as shown in SEQ ID NO.2, or an amino acid sequence modified by a certain degree of amino acid substitution, insertion, substitution, addition, deletion, etc. on the basis of SEQ ID NO.2, or an amino acid sequence having more than 80% homology with the amino acid sequence of SEQ ID NO.2;

[0014] The amino acid sequence of COL17A3b is as shown in SEQ ID NO.3, or an amino acid sequence modified by a certain degree of amino acid substitution, insertion, substitution, addition, deletion, etc. on the basis of SEQ ID NO.3, or an amino acid sequence having more than 80% homology with the amino acid sequence of SEQ ID NO.3;

[0015] The amino acid sequence of COL17A3c is as shown in SEQ ID NO.4, or an amino acid sequence modified by amino acid substitution, insertion, substitution, addition, deletion, etc. to a certain extent on the basis of SEQ ID NO.4, or an amino acid sequence having a homology greater than 80% with the amino acid sequence of SEQ ID NO.4;

[0016] The amino acid sequence of COL17A3d is as shown in SEQ ID NO.5, or an amino acid sequence modified by amino acid substitution, insertion, substitution, addition, deletion, etc. to a certain extent on the basis of SEQ ID NO.5, or an amino acid sequence having a homology greater than 80% with the amino acid sequence of SEQ ID NO.5;

[0017] The amino acid sequence of COL17A3e is as shown in SEQ ID NO.6, or an amino acid sequence modified by amino acid substitution, insertion, substitution, addition, deletion, etc. to a certain extent on the basis of SEQ ID NO.6, or an amino acid sequence having a homology greater than 80% with the amino acid sequence of SEQ ID NO.6.

[0018] Furthermore, a protein tag is added to the N-terminus of the collagen, preferably a Trx-his-thrombin-Stag tag at the N-terminus.

[0019] The second aspect of the present invention provides a method for preparing recombinant human type XVII collagen, which method comprises the following steps:

[0020] 1) Retrieve the amino acid sequence of type XVII collagen with the sequence number: Q9UMD9.3 from the protein database NCBI. Its amino acid sequence is as shown in SEQ ID NO.1. Use the NCIB BLAST tool to find the conserved regions in the sequence, use the MODELLER and SWISS-MODEL tools to predict the three-dimensional structure of the collagen, use molecular dynamics simulation to optimize its structure and perform energy minimization research, and predict the potential active functional regions through a molecular docking tool: the amino acid sequences shown in SEQ ID NO.2-6;

[0021] 2) Clone the amino acid sequences shown in SEQ ID NO.2 - 6 into the E. coli expression vector pET32a; construct the 5 target fragments into the pET32a plasmid through the BglII and EcoRI restriction enzyme sites, and connect Trx, his, thrombin, and S tag at the N - terminus of the target fragments, while adding an enterokinase cleavage site to construct the expression plasmids pET32a - A3a, pET32a - A3b, pET32a - A3c, pET32a - A3d, and pET32a - A3e; transform the expression plasmids into the competent cell BL21(DE3) by heat shock method and culture to obtain the bacterial cells.

[0022] 3) Resuspend the bacterial cells obtained in step 2) with the ultrasonic disruption buffer, then perform ultrasonic disruption to lyse the bacterial cells. After disruption, use a refrigerated centrifuge to remove the precipitate, take the supernatant to a new centrifuge tube and centrifuge again, retaining the supernatant; connect the AKTA pipeline, set the flow rate at 8 mL / min, and rinse with 0.5 M NaOH; then rinse with ddH2O; install the purification column, rinse it, and then load the centrifuged supernatant with a flow rate of 4 ml / min; wash away impurities with 8 mM imidazole, and then perform linear gradient elution with different concentrations of imidazole, collect the eluate, and dialyze it overnight into PBS; for the target protein that needs to remove the tag, add enterokinase with His - tag, incubate at 4°C for 16 h, and collect the flow - through solution, which is the tag - removed target collagen COL17A3a, COL17A3b, COL17A3c, COL17A3d, COL17A3e.

[0023] Furthermore, the specific steps of the culture in step 2) are as follows: Take the BL21 competent cells, dissolve them on ice, add the expression plasmid, mix well and let it stand on ice, then perform heat shock in a water bath. After adding the non - resistant 2YT medium, culture and resuscitate at 37°C, 200 rpm on a shaker for 60 min; spread the bacterial solution on the LB solid medium containing Amp resistance and culture overnight in a 37°C incubator; then pick a single - clone colony and inoculate it into 1000 mL of the medium, culture at 37°C, 250 rpm until the OD600 reaches 0.8, add 1 mM IPTG to induce expression for 18 h, and then centrifuge with a refrigerated centrifuge to collect the bacterial cells.

[0024] Furthermore, the specific method for rinsing the purification column in step 3) is as follows: Set the flow rate at 8 mL / min, rinse with 60 mL of ddH2O, then rinse with 80 mL of 0.1 M NaOH; then rinse with 80 mL of ddH2O; finally, rinse with 50 mL of 0.01 M PBS before loading the sample.

[0025] The third aspect of the present invention provides a product, which is the collagen prepared by the above-mentioned collagen or the above-mentioned preparation method or a composition. The product includes drugs, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, and cosmetics.

[0026] The fourth aspect of the present invention provides the application of the above-mentioned collagen or the collagen prepared by the above-mentioned preparation method in the preparation of drugs, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, and cosmetics for treating skin anti-aging.

[0027] The fifth aspect of the present invention provides the application of the above-mentioned collagen or the collagen prepared by the above-mentioned preparation method in the preparation of drugs, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, and cosmetics for treating hair loss and white hair prevention.

[0028] Collagen type XVII has an extremely large molecular weight (180KD) and contains 1497 amino acids. However, little is known about its active functional regions. Moreover, collagen type XVII is a transmembrane protein, and it is difficult to produce using traditional collagen extraction methods, with extremely high costs. Additionally, the presence of its transmembrane domain prevents it from being effectively secreted extracellularly, making it difficult to achieve mass production.

[0029] The present invention uses computer simulation and molecular docking methods to study the three-dimensional spatial structure of collagen type XVII, and uses artificial intelligence methods to predict the functional fragments of collagen type XVII, reducing the amino acid sequence of collagen from 1497 to an extracellular functional region within 200AA. This not only reduces its molecular weight, enabling its secretory expression, but also results in high production yields, short production cycles, and low costs using the Escherichia coli expression system, making it easy to carry out large-scale preparation. Additionally, compared with traditional collagen and macromolecular recombinant collagen.

[0030] The present invention uses the Escherichia coli recombinant expression system to recombinantly express the selected collagen type XVII, constructing the target sequences A3a, A3b, A3c, A3d, A3e into the prokaryotic expression vector PET32a. For convenient product purification, tag sequences such as Trx, His, Thrombin, and S tag are fused to the N-terminus of the A3a, A3b, A3c, A3d, A3e sequences. Through prokaryotic (BL21) fermentation, the target sequences are expressed, and then the supernatant is taken for nickel column affinity purification. The tagged candidate sequences are obtained. If further tag removal is required, enterokinase can be used for excision to obtain the target collagen sequence.

[0031] Through software analysis, the present invention locks the functional region of collagen within 200 AA, and performs prokaryotic expression and purification on its functional fragments to obtain the target collagen fragments A3a, A3b, A3c, A3d, and A3e. The present invention proves that these four fragments have good biological activity through adhesion activity identification in keratinocytes HACAT.

[0032] Amino acid sequence of type XVII collagen: (SEQ ID NO.1)

[0033]

[0034] A3a sequence: (SEQ ID NO.2) KGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEA.

[0035] A3b sequence: (SEQ ID NO.3)

[0036] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVG.

[0037] A3c sequence: (SEQ ID NO.4)

[0038] KGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVG.

[0039] A3d sequence: (SEQ ID NO.5)

[0040] DRGFPGTPGIPGPLGHPGPQGPKGQKGSVG.

[0041] A3e sequence: (SEQ ID NO.6)

[0042] GIPGPLGHPGPQGPKGQKGSVGDPGMEGPM. Description of the Drawings

[0043] Figure 1Electrophoresis diagram of COL17A3a fusion protein with Trx-his-thrombin-S tag label;

[0044] Figure 2 Electrophoresis diagram of COL17A3b fusion protein with Trx-his-thrombin-S tag label;

[0045] Figure 3 Electrophoresis diagram of COL17A3c fusion protein with Trx-his-thrombin-S tag label;

[0046] Figure 4 Electrophoresis diagram of COL17A3d fusion protein with Trx-his-thrombin-S tag label;

[0047] Figure 5 Electrophoresis diagram of COL17A3e fusion protein with Trx-his-thrombin-S tag label;

[0048] Figure 6 Adhesion activity assay diagram of COL17A3a, COL17A3b, COL17A3c, COL17A3d, COL17A3e;

[0049] Figure 7 Proliferation activity assay diagram of COL17A3a, COL17A3b, COL17A3c, COL17A3d, COL17A3e. Detailed implementation mode

[0050] The present invention will be further described below in conjunction with the accompanying drawings of the specification for better understanding of the technical solution.

[0051] Example 1: Construction, expression and purification of recombinant type XVII collagen. Retrieve the amino acid sequence of type XVII collagen from the protein database (NCBI), and the sequence number is: Q9UMD9.3 (SEQ ID NO.1). Use the NCIB BLAST tool to find the conserved regions in the sequence. Then use tools such as MODELLER and SWISS-MODEL to predict the three-dimensional structure of collagen. Use molecular dynamics simulation to optimize its structure and study energy minimization, and predict potential active sites through molecular docking tools.

[0052] Five potential active functional regions (SEQ ID NO.2 - NO.6) are designed according to the prediction results of the present invention.

[0053] 1. Construction and Expression: The amino acid sequences shown in SEQ ID NO.2 - NO.6 were cloned into the Escherichia coli expression vector pET32a. Five target fragments were constructed into the pET32a plasmid through the BglII and EcoRI restriction sites. For convenient later expression and purification, the Trx, his, thrombin, and S tag were linked to the N - terminus of the target fragments, and the enterokinase cleavage site was added for later tag removal of the samples. The expression plasmids of A3a, A3b, A3c, A3d, and A3e were constructed. The expression plasmids were transformed into the competent cell BL21(DE3) by heat shock method. Take the BL21 competent cells, dissolve them on ice, add the expression plasmid, mix well and let it stand on ice for 30 min, then heat shock in a 42°C water bath for 90 s, add 900 μl of antibiotic - free 2YT medium, and culture at 37°C and 200 rpm on a shaker for 60 min. Then take about 50 μl of the bacterial solution and spread it on the LB solid medium (containing Amp resistance). Culture it in a 37°C incubator overnight. The next day, pick a monoclonal colony and inoculate it into 1000 mL of the medium, and culture at 37°C and 250 rpm until the OD 600 reaches about 0.8, add 1 mM IPTG to induce expression for 18 h. Then use a refrigerated centrifuge to centrifuge and collect the bacterial cells.

[0054] 2. Purification: Resuspend the bacterial cells with the ultrasonic disruption buffer, and then perform ultrasonic disruption to lyse the bacterial cells (240 W, 60 min). After disruption, use a refrigerated centrifuge to centrifuge (centrifuge at 16000 rpm for 20 min) to remove the precipitate. Take the supernatant and transfer it to a new centrifuge tube, centrifuge again at 16000 rpm for 40 min, and retain the supernatant. Connect the AKTA pipeline, set the flow rate to 8 mL / min, and rinse with 0.5 M NaOH for 2 h; then rinse with ddH2O for 2 h. Install the purification column, set the flow rate to 8 mL / min, rinse with ddH2O for 60 mL, then rinse with 0.1 M NaOH for 80 mL; rinse with ddH2O for 80 mL again; finally, rinse with 0.01 M PBS for 50 mL before loading the sample, and then load the centrifuged supernatant with a flow rate of 4 mL / min; wash away impurities with 8 mM imidazole, and then perform linear gradient elution with different concentrations of imidazole. Collect the eluate and dialyze it overnight into PBS. For the target protein that needs to remove the tag, an appropriate amount of enterokinase with His - tag can be added, incubated at 4°C for 16 h, and collect the flow - through fraction, which is the tag - removed target collagen COL17A3a, COL17A3b, COL17A3c, COL17A3d, COL17A3e.

[0055] The electrophoresis pattern of the COL17A3a fusion protein with Trx - his - thrombin - S tag is asFigure 1 As shown; the theoretical molecular weight of the tagged COL17A3a is 35.4 KD, and the apparent molecular weight by SDS-PAGE is approximately 43 KD.

[0056] The electrophoresis pattern of the COL17A3b fusion protein with the Trx-his-thrombin-S tag is as Figure 2 shown; the theoretical molecular weight of the tagged COL17A3b is 34.3 KD, and the apparent molecular weight by SDS-PAGE is approximately 40 KD.

[0057] The electrophoresis pattern of the COL17A3c fusion protein with the Trx-his-thrombin-S tag is as Figure 3 shown; the theoretical molecular weight of the tagged COL17A3c is 33.1 KD, and the apparent molecular weight by SDS-PAGE is approximately 35 KD.

[0058] The electrophoresis pattern of the COL17A3d fusion protein with the Trx-his-thrombin-S tag is as Figure 4 shown; the theoretical molecular weight of the tagged COL17A3d is 19.7 KD, and the apparent molecular weight by SDS-PAGE is approximately 22 KD.

[0059] The electrophoresis pattern of the COL17A3e fusion protein with the Trx-his-thrombin-S tag is as Figure 5 shown. The theoretical molecular weight of the tagged COL17A3e is 19.7 KD, and the apparent molecular weight by SDS-PAGE is approximately 22 KD.

[0060] Example 2: Determination of the adhesion activity of COL17A3a, COL17A3b, COL17A3c, COL17A3d, and COL17A3e

[0061] The concentration of the collagen to be tested was detected by the ultraviolet absorption method. The specific measurement method is as follows: The ultraviolet light absorption of the sample was measured at 215 nM and 225 nM respectively, and then the concentration of collagen was calculated using the empirical formula C (mg / mL) = 0.144 * (A215 - A225), where A215 < 1.5.

[0062] The collagen was diluted with PBS to a concentration of 0.3 mg / mL; 100 uL was added to each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate and coated overnight at 4 °C. The next day, the coating solution was aspirated with a pipette, and the ELISA plate was washed 2 times with PBS; then 200 uL of 2% BSA was added to each well. Incubate at 37 °C for 1 h; then remove the coating solution, wash the ELISA plate 2 times with PBS, and add 10 5HACAT cells (DMEM + 10% FBS) were incubated at 37 °C for an additional 2 h, then the cell culture medium was aspirated, and each well was washed 3 times with PBS. Finally, 100 μL of 10% CCK8 detection reagent (Beyotime, C0042) was added to each well. After incubating at 37 °C in the dark for 1 - 3 h, the OD450 value was read. The strength of the OD450 signal can directly reflect the number of adherent cells.

[0063] The results are shown in Figure 6 . COL17 A3a, COL17 A3b, COL17 A3c, COL17 A3d, and COL17 A3e all exhibited good cell adhesion activity.

[0064] Example 3: Proliferation activity assay of COL17A3a, COL17 A3b, COL17 A3c, COL17 A3d, and COL17 A3e

[0065] Resuscitate HACAT cells and culture them in DMEM + 10% FBS medium, passaging once every 3 - 4 days. When the cell viability reaches over 95%, perform the collagen proliferation activity assay.

[0066] Digest HACAT cells with trypsin, resuspend them in serum-free medium after digestion, gently disperse the cells, then count and adjust the cell density to 1E5 cells / mL, and add 100 μL per well to a 96-well cell culture plate;

[0067] Then dilute the collagen to be tested with serum-free DMEM to a concentration of 0.5 mg / mL and add 100 μL per well to the 96-well cell culture plate. Incubate in a 37 °C, 5% CO2 incubator for 72 h. Then aspirate the culture supernatant, add DMEM serum-free medium containing 10% CCK8 to each well, and continue to incubate for 1 - 3 h. Read the OD450 value. The signal intensity of OD450 can directly reflect the proliferation of the cells.

[0068] The results are shown in Figure 7 . COL17 A3a, COL17 A3b, COL17 A3c, COL17 A3d, and COL17 A3e all exhibited good cell proliferation activity.

Claims

1. A recombinant human type XVII collagen, characterized in that, The recombinant human type XVII collagen is: COL17A3d, and the amino acid sequence of COL17A3d is as shown in SEQ ID NO.

5.

2. A recombinant human type XVII collagen according to claim 1, characterized in that, A protein tag is added to the N-terminus of the collagen, and the protein tag is a Trx-his-thrombin-S tag.

3. The preparation method of a recombinant human type XVII collagen according to any one of claims 1-2, characterized in that, It includes the following steps: 1) Retrieve the amino acid sequence of type XVII collagen with the sequence number Q9UMD9.3 from the protein database NCBI. Its amino acid sequence is as shown in SEQ ID NO.

1. Use the NCIB BLAST tool to find the conserved regions in the sequence. Use the MODELLER and SWISS-MODEL tools to predict the three-dimensional structure of the collagen. Use molecular dynamics simulation to optimize its structure and perform energy minimization research, and predict the potential active functional regions through molecular docking tools: the amino acid sequence shown in SEQ ID NO.5; 2) Clone the amino acid sequence shown in SEQ ID NO.5 into the Escherichia coli expression vector PET32a; construct the target fragment into the PET32a plasmid through the BglII and EcoRI restriction enzyme sites, and connect Trx, his, thrombin, and S tag at the N-terminus of the target fragment. At the same time, add an enterokinase cleavage site to construct the PET32a-Trx-his-thrombion-S-A3d expression plasmid; transform the expression plasmid into the competent cell BL21(DE3) by heat shock method and culture to obtain the bacterial cells; 3) Resuspend the bacterial cells obtained in step 2) with ultrasonic lysis buffer, then perform ultrasonic lysis of the bacterial cells. After lysis, use a refrigerated centrifuge to remove the precipitate. Take the supernatant to a new centrifuge tube and centrifuge again, retaining the supernatant; connect the AKTA pipeline, set the flow rate to 8 mL / min, and rinse with 0.5 M NaOH; then rinse with ddH2O; install the purification column and rinse, then load the centrifuged supernatant, with a flow rate of 4 min / min; wash away impurities with 8 mM imidazole, and then elute with a linear gradient of different concentrations of imidazole, collect the eluate, and dialyze overnight into PBS; For the target protein that needs to remove the tag, add enterokinase with a His tag, incubate at 4°C for 16 h, and collect the flow-through fraction, which is the target collagen COL17A3d without the tag.

4. The preparation method of a recombinant human type XVII collagen according to claim 3, characterized in that The specific steps of the culture in step 2) are as follows: Take BL21 competent cells, dissolve them on ice, add the expression plasmid, mix well and let it stand on ice, then perform heat shock in a water bath. After adding the non-resistant 2YT medium, culture and resuscitate at 37°C and 200 rpm for 60 min; spread the bacterial solution on the LB solid medium containing Amp resistance and culture overnight in a 37°C incubator; then pick a monoclonal colony and inoculate it into 1000 mL of the medium, culture at 37°C and 250 rpm until the OD600 reaches 0.8, add 1 mM IPTG to induce expression for 18 h, and then centrifuge with a refrigerated centrifuge to collect the bacterial cells.

5. The preparation method of a recombinant human type XVII collagen according to claim 3, characterized in that The specific method for purifying column flushing in step 3) is as follows: set the flow rate at 8 mL / min, flush with ddH2O for 60 mL, then flush with 0.1 M NaOH for 80 mL; then flush with ddH2O for 80 mL; finally, flush with 0.01 M PBS for 50 mL before loading the sample.

6. A product, the product comprising the collagen according to any one of claims 1-2 or the collagen prepared by the preparation method according to any one of claims 3-5, the product including drugs, medical devices, biomaterials, tissue engineering products, cosmetics.

7. Use of the product according to claim 6 in the preparation of drugs, medical devices, biomaterials, tissue engineering products, cosmetics for treating skin anti-aging and tissue repair.

8. Use of the product according to claim 6 in the preparation of drugs, medical devices, biomaterials, tissue engineering products, cosmetics for treating hair loss and gray hair.

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