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

Abstract

The invention relates to a recombinant human XVII type collagen as well as a preparation method and application of the recombinant human XVII type collagen. The recombinant human XVII type collagen is any one of COL17A 3a, COL17A 3b, COL17A 3c, COL17A 3d or COL17A 3e. According to the invention, the functional fragments of the XVII collagen are analyzed by using computer software, the minimum functional region of the XVII collagen is screened, and expression and extraction are carried out by using an escherichia coli recombinant expression system, so that the target functional fragments with higher yield can be obtained.

Description

Technical Field

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

[0002] Collagen is the earliest discovered and most abundant type of extracellular matrix protein, and is a key structural component of the extracellular matrix. It is widely found in skin, bones, tendons, teeth, internal organs (such as stomach, intestines, heart and lungs, blood vessels and esophagus), ligaments, sclera, eyes and other tissues and organs, accounting for 30%-40% of the human body. 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 three or more levels of structure have physiological functions, and 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 composed of GXY (X, Y represent any amino acid residue other than Gly, X is often Pro, and Y is often Hyp) repeating units. The most common sequence is the "glycine-proline-hydroxyproline" sequence, which accounts for about 12% of the entire "GXY" sequence, "glycine-proline-Y" or "glycine-X-hydroxyproline" sequences account for about 44%, and other "glycine-XY" sequences make up the remaining 44%. The number of these repeated tripeptides "glycine-XY" is close to 1 / 3 of the total sequence, which plays 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 superhelical manner with the same axis as the center. Collagen plays an important role in organ support and body function protection. It is also the main factor in the toughness of connective tissue. Connective tissue adheres cells throughout the body and connects them into tissues and organs. Therefore, collagen plays an important role in the human body.

[0003] 28 different types of collagen have been discovered so far. Collagen in different races and tissues has different chemical compositions or different configurations. According to the function of collagen, it can be divided into fibroblastic collagen and non-fibroblastic collagen. From the human body, the most common collagen consists of four forms: Type I collagen is the largest in the human body and mainly exists in adult skin and bone tissue, Type II collagen mainly exists in cartilage tissue, and Type III collagen mainly exists in infants' skin, blood vessels, endothelium, intestines, and stomach organs. Type IV collagen mainly exists in the basement membrane, placenta, and corpus callosum of various tissues and organs.

[0004] Type XVII collagen (Collagen XVII, type 17 collagen, COL17) is a transmembrane protein. It was first discovered by Diaz et al. in bullous pemphigoid (BP) in the 1980s. It is a type II transmembrane protein. Its most obvious structural feature is that the N-terminus is located in the cell and consists of 501 amino acids, including 4 tandem repeats of 24-26 amino acids. The C-terminus is a longer extracellular peptide bond that connects to the anchor fibers of the transparent plate of the basement membrane of the skin. The extracellular collagen domain of type XVII collagen contains repeated "glycine-XY" triads, where X is usually proline and Y is hydroxyproline. The interspersed non-collagenous domains are called NC1, NC2, and up to NC16 from the carboxyl terminus (C-terminus). The higher-order structure of type XVII collagen is a trimer driven by the triple helical assembly of the collagen domain. Immunoelectron microscopy studies have shown that in the basement membrane, the extracellular domain (ECD) of type XVII collagen passes through the lamina lucida of the basement membrane into the lamina compacta and then bends back into the lamina lucida. Rotational shadow electron microscopy imaging has also demonstrated that the extracellular domain (ECD) has a rod-shaped morphology, while the basic intracellular domain (ICD) is spherical.

[0005] Type XVII collagen is a structural component of hemidesmosomes and is abundantly expressed in skin epithelial cells and other stratified epithelial cells, such as the oral mucosa, cornea, cervix, and vagina. Type XVII collagen is also abundant in squamous epithelial cells, syncytial cells, and cytotrophoplastic cells of the placenta. Type XVII collagen in the mouth is essential for the formation of tooth enamel. Type XVII collagen is expressed at lower levels in mammary and intestinal epithelia. In stratified epithelium, type XVII collagen is mainly expressed in basal cells. In the skin, it is present in both hair follicles and in the interstitial epidermis. In addition to hemidesmosomal expression, non-hemidesmosomal type XVII collagen is also present on the apical surface of the soluble membrane of basal keratinocytes. Hemidesmosomes are the main connecting structure between basal cells and the underlying basement membrane zone, which are formed by the interlocking of irregular protrusions of the dermal cell membrane of keratinocytes and the basement membrane zone. Hemidesmosomes have the function of anchoring cells to the basement membrane, thereby strengthening the connection between cells. Hemidesmosomes decrease with age and cause microseparation of basal cells. Among all hemidesmosome components, type XVII collagen is the only one that is significantly reduced during aging. Current studies have also shown that the instability of hemidesmosomes is caused by the hydrolysis of type XVII collagen caused by genomic instability caused by intrinsic aging and ultraviolet-induced photoaging. Irradiation with UVB can cause photoaging of the skin, and pigmentation and hemidesmosome damage can occur in both mouse and human epidermis. The skin pigmentation of mice lacking type XVII collagen is more obvious and wound healing is slow; while overexpression of type XVII collagen can significantly reduce pigmentation and promote wound healing.

[0006] Type XVII collagen plays an important role in maintaining the connection between intracellular and extracellular structural elements of epidermal cell adhesion. The extracellular domain (ECD) of type XVII collagen directly binds to integrin α6, laminin 332, and collagen IV. These interactions mediate the adhesion of keratinocytes to the basal matrix and regulate keratinocyte migration together with the shedding of the extracellular domain (ECD). In addition, studies have shown that type XVII collagen may bind to or interact with periplakin, DFNB31, CSTF2T, PPLIL1, PLOD3, CDH1, PNKP, HRAS, SPDL1, EGFR, and ubiquilin2. These interactors may be involved in the biosynthesis, transport, and post-translational modification of type XVII collagen 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 niche of stem cells in the skin (such as epidermal stem cells, hair follicle stem cells, and melanocyte stem cells) is essential for the function of stem cells. Adhesion between cells and between cells and the extracellular matrix (ECM) is essential for establishing and maintaining the stem cell niche structure. Adhesion to the underlying extracellular matrix is ​​considered to be an important factor in maintaining epidermal stem cells (ESCs). Type XVII collagen plays an important role in maintaining the stem cell niche. Therefore, type XVII collagen 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 (HFSC) are responsible for the periodic 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. As hair follicle stem cells age, the DNA damage response (DDR) is prolonged, which can activate ELANE (neutrophil elastase: an enzyme present in neutrophils, involved in immune response and inflammatory process, and capable of degrading a variety of proteins), leading to the hydrolysis and loss of collagen COL17, a key molecule for maintaining HFSCs, thereby triggering the aging of HFSCs. Aged hair follicle stem cells (HFSC) are terminally differentiated into epidermal keratinocytes through the epidermis and periodically cleared from the skin. With the repetition of this atypical division, hair follicle stem cells (HFSC) separate from the basement membrane, the hair follicle stem cell niche gradually shrinks, and the hair follicle itself becomes smaller (miniaturized), eventually leading to sparse hair and shedding. Studies have shown that mandatory maintenance of collagen COL17 expression can inhibit hair follicle stem cell aging, thereby delaying excessive hair loss.

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

[0010] Although the efficacy of type XVII collagen in the field of anti-aging and anti-hair loss has been confirmed, its main core functional fragments are little known. The present invention uses computer simulation and artificial intelligence to find the minimum active functional fragment of type XVII collagen, and then uses a recombinant expression system to express the active fragment, and finds 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] In view of the problems existing in the prior art, the purpose of the present invention is to provide a recombinant human type XVII collagen and a preparation method and application thereof, which are specifically achieved through the following technical solutions:

[0012] A first aspect of the present invention provides a recombinant human type XVII collagen, wherein 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 amino acid substitution, insertion, replacement, addition, deletion, etc. to a certain extent on the basis of SEQ ID NO.2, or an amino acid sequence having greater 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 amino acid substitution, insertion, replacement, addition, deletion, etc. to a certain extent on the basis of SEQ ID NO.3, or an amino acid sequence having greater 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 to a certain extent by amino acid substitution, insertion, replacement, addition, deletion, etc. on the basis of SEQ ID NO.4, or an amino acid sequence having greater than 80% homology 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, replacement, addition, deletion, etc. to a certain extent on the basis of SEQ ID NO.5, or an amino acid sequence having greater than 80% homology 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 to a certain extent by amino acid replacement, insertion, substitution, addition, deletion, etc. on the basis of SEQ ID NO.6, or an amino acid sequence having greater than 80% homology 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 is added to the N-terminus of the collagen.

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

[0020] 1) The amino acid sequence of XVII collagen was retrieved from the protein database NCBI, and the sequence number was Q9UMD9.3. The amino acid sequence was shown in SEQ ID NO.1. The conserved region in the sequence was searched using the NCIB BLAST tool. The three-dimensional structure of collagen was predicted using the MODELLER and SWISS-MODEL tools. The structure was optimized and the energy was minimized using molecular dynamics simulation. The potential active functional region was predicted using the molecular docking tool: the amino acid sequence shown in SEQ ID NO.2 to 6;

[0021] 2) The amino acid sequences shown in SEQ ID NO.2 to 6 were cloned into the Escherichia coli expression vector PET32a; the fragments of the five items were constructed into the PET32a plasmid through the BglII and EcoRI restriction sites, and Trx, his, thrombin and S tag tags were connected to the N-terminus of the target fragment, and an enterokinase restriction site was added to construct PET32a-A3a, PET32a-A3b, PET32a-A3c, PET32a-A3d, and PET32a-A3e expression plasmids; the expression plasmids were transformed into competent cells BL21 (DE3) by the heat shock method and cultured to obtain bacteria;

[0022] 3) Resuspend the bacteria obtained in step 2) with ultrasonic disruption buffer, then ultrasonically disrupt and lyse the bacteria, remove the precipitate by centrifugation in a refrigerated centrifuge after disruption, take the supernatant into a new centrifuge tube and centrifuge again, and retain the supernatant; connect the AKTA pipeline, set the flow rate to 8 mL / min, rinse with 0.5 M NaOH; then rinse with ddH2O; install the purification column and rinse, then load the supernatant after centrifugation, with a flow rate of 4 ml / min; wash with 8 mM imidazole, then elute with a linear gradient of imidazole of different concentrations, collect the eluate, and dialyze into PBS overnight; if the target protein with a tag needs to be removed, add enterokinase with a His tag, incubate at 4°C for 16 hours, and collect the flow-through, which is the target collagen COL17A3a, COL17A3b, COL17A3c, COL17A3d, COL17A3e with the tag removed.

[0023] Further, the specific steps of culturing in step 2) are: take BL21 competent cells, dissolve them on ice, add the expression plasmid, mix them and let them stand on ice, then heat shock them in a water bath, add 2YT medium without resistance, and culture them on a shaker at 37°C and 200rpm for 60 minutes; take the bacterial solution and spread it on LB solid medium containing Amp resistance, and culture it in a 37°C incubator overnight; then pick a monoclonal colony and inoculate it into 1000mL of culture medium, culture it at 37°C and 250rpm until OD600 reaches 0.8, add 1mM IPTG to induce expression for 18 hours, and then centrifuge it in a refrigerated centrifuge to collect the bacteria.

[0024] Furthermore, the specific method for flushing the purification column in step 3) is: set the flow rate to 8 mL / min, flush 60 mL with ddH2O, then flush 80 mL with 0.1 M NaOH; then flush 80 mL with ddH2O; and finally flush 50 mL with 0.01 M PBS before loading.

[0025] The third aspect of the present invention provides a product, which is the collagen mentioned above or the collagen or composition prepared by the above preparation method. The product includes a drug, a drug composition, a medical device, a biomaterial, a tissue engineering product, a cosmetic or a health product.

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

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

[0028] Type XVII collagen has a very large molecular weight (180KD) and contains 1497 amino acids, but very little is known about its active functional area. Type XVII collagen is a transmembrane protein, and the traditional collagen extraction method is difficult and costly to produce. In addition, the presence of its transmembrane domain prevents it from being effectively secreted outside the cell, making it difficult to mass produce.

[0029] The present invention uses computer simulation and molecular docking to study the three-dimensional structure of type XVII collagen, uses artificial intelligence to predict the functional fragments of type XVII collagen, and reduces the amino acid sequence of collagen from 1497 to an extracellular functional region within 200AA. The molecular weight is reduced, which not only enables secretory expression, but also uses an Escherichia coli expression system to produce high expression yield, short production cycle, and low cost, and is easy to prepare on a large scale. In addition, compared with traditional collagen and macromolecular recombinant collagen.

[0030] The present invention utilizes the recombinant expression system of Escherichia coli to recombinantly express the selected XVII collagen, and constructs the target sequence A3a, A3b, A3c, A3d, and A3e into the prokaryotic expression vector PET32a. In order to facilitate product purification, the N-terminal of the A3a, A3b, A3c, A3d, and A3e sequences are fused with tag sequences such as Trx, His, Thrombin, and S tag. The target sequence is expressed by prokaryotic (BL21) fermentation, and then the supernatant is taken for nickel column affinity purification. The candidate sequence with the tag is obtained. If the tag needs to be further removed, it can be removed by enterokinase to obtain the target collagen sequence.

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

[0032] Type XVII collagen amino acid sequence: (SEQ ID NO.1)

[0033] MDVTKKNKRDGTEVTERIVTETVTTRLTSLPPKGGTSNGYAKTAS

[0034] LGGGSRLEKQSLTHGSSGYINSTGSTRGHASTSSYRRAHSPASTLP

[0035] NSPGSTFERKTHVTRHAYEGSSSGNSSPEYPRKEFASSSTRGRSQT

[0036] RESEIRVRLQSASPSTRWTELDDVKRLLKGSRSASVSPTRNSSNTL

[0037] PIPKKGTVETKIVTASSQSVSGTYDATILDANLPSHVWSSTLPAGS

[0038] SMGTYHNNMTTQSSSLLNTNAYSAGSVFGVPNNMASCSPTLHPG

[0039] LSTSSSVFGMQNNLAPSLTTLSHGTTTSTAYGVKKNMPQSPAAV

[0040] NTGVSTSAACTTSVQSDDLLHKDCKFLILEKDNTPAKKEMELLIM

[0041] TKDSGKVFTASPASIAATSFSEDTLKKEKQAAYNADSGLKAEANG

[0042] DLKTVSTKGKTTTADIHSYGSSGGGGSGGGGGVGGAGGGPWGP

[0043] APAWCPCGSCCSWWKWLLGLLLTWLLLLGLLFGLIALAEEVRKL

[0044] KARVDELERIRRSILPYGDSMDRIEKDRLQGMAPAAGADLDKIGL

[0045] HSDSQEELWMFVRKKLMMEQENGNLRGSPGPKGDMGSPGPKGD

[0046] RGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREG

[0047] PMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGS

[0048] PGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEK

[0049] GPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQG

[0050] LTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGKIKGEPGA

[0051] PGKIVTSEGSSMLTVPGPPPGPGAMGPPGPGPGAGPAGLPGH

[0052] QEVLNLQGPPPGPGPRGPPGPSIPGPPGPRGPPGEGLPGPPGPPGGSF

[0053] LSNSETFLSGPPGPPPGPGPKGDQGPPPGPRGHQGEQGLPGFSTSGS

[0054] SSFGLNLQGPPPGPGPQGPKGDKGDPGVPPGALGIPSGPSEGGSSST

[0055] MYVSGPPGPPPGPPGSISSSGQEIQQYISEYMQSDSIRSYLSGVQ

[0056] GPPGPPPGPGPVTTTITGETFDYSELASHVVSYLRTSGYGVSLFSSSI

[0057] SSEDILAVLQRDDVRQYLRQYLMGPRGPPPGPGASGDGSLLSLDY

[0058] AELSSRILSYMSSSGISIGLPGPPGPPGLPGTSYEELLSLLRGSEFRGI

[0059] VGPPGPPGPPGIPGNVWSSISVEDLSSYLHTAGLSFIPGPPGPPGPP

[0060] GPRGPPGVSGALATYAAENSDSFRSELISYLTSPDVRSFIVGPPGPP

[0061] GPQGPPGDSRLLSTDASHSRGSSSSSHSSSVRRGSSYSSSMSTGGG

[0062] GAGSLGAGGAFGEAAGDRGPYGTDIGPGGGYGAAAEGGMYAG

[0063] NGGLLGADFAGDLDYNELAVRVSESMQRQGLLQGMAYTVQGPP

[0064] GQPGPQGPPGISKVFSAYSNVTADLMDFFQTYGAIQGPPGQKGEM

[0065] GTPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQVYAGRRRRRSIAVKP.

[0066] A3a sequence: (SEQ ID NO.2)

[0067] KGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRG

[0068] REGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPG

[0069] PQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGD

[0070] MGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEA。

[0071] A3b sequence: (SEQ ID NO.3)

[0072] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSV

[0073] GDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRG

[0074] FPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPM

[0075] GPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVG.

[0076] A3c sequence: (SEQ ID NO.4)

[0077] KGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRG

[0078] REGPMGPRGEAGSGPPKGDMGSPGPKGDRGFPGTPGIPGPLGHPG

[0079] PQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVG.

[0080] A3d sequence: (SEQ ID NO.5)

[0081] DRGFPGTPGIPGPLGHPGPQGPKGQKGSVG.

[0082] A3e sequence: (SEQ ID NO.6)

[0083] GIPGPLGHPGPQGPKGQKGSVGDPGMEGPM. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Electrophoresis of COL17A3a fusion protein with Trx-his-thrombin-S tag;

[0085] Figure 2 This is the electrophoresis of COL17A3b fusion protein with Trx-his-thrombin-S tag;

[0086] Figure 3This is the electrophoresis of COL17A3c fusion protein with Trx-his-thrombin-S tag;

[0087] Figure 4 This is the electrophoresis of COL17A3d fusion protein with Trx-his-thrombin-S tag;

[0088] Figure 5 This is the electrophoresis of COL17A3e fusion protein with Trx-his-thrombin-S tag;

[0089] Figure 6 It is a graph of the adhesion activity assay of COL17A3a, COL17 A3b, COL17 A3c, COL17 A3d, and COL17A3e;

[0090] Figure 7 This is a graph showing the proliferation activity assay of COL17A3a, COL17 A3b, COL17 A3c, COL17 A3d, and COL17A3e. DETAILED DESCRIPTION

[0091] The present invention is further described below in conjunction with the accompanying drawings in order to better understand the technical solution.

[0092] Example 1: Construction, expression and purification of type XVII recombinant collagen The amino acid sequence of type XVII collagen was retrieved from the protein database (NCBI), and the sequence number was Q9UMD9.3 (SEQ ID NO.1. The conserved region in the sequence was searched using the NCIB BLAST tool. Then, the three-dimensional structure of the collagen was predicted using tools such as MODELLER and SWISS-MODEL. The structure was optimized and the energy was minimized using molecular dynamics simulation, and potential active sites were predicted using molecular docking tools.

[0093] The prediction results of the present invention designed 5 potential active functional regions (SEQ ID NO.2-NO.6).

[0094] 1. Construction and expression: The amino acid sequences shown in SEQ ID NO.2-NO.6 were cloned into the E. coli expression vector PET32a. The fragments of the five entries were constructed into the PET32a plasmid through the BglII and EcoRI restriction sites. In order to facilitate the later expression and purification, the target fragments were connected to the N-terminus of the target fragments with Trx, his, thrombin and S tag tags, and the enterokinase restriction site was added. The tags were removed from the samples later. Construct A3a, A3b, A3c, A3d, and A3e expression plasmids. The expression plasmids were transformed into competent cells BL21 (DE3) by heat shock method. The BL21 competent cells were taken, dissolved on ice, and the expression plasmids were added. After mixing, they were placed on ice for 30 minutes, and then heat-shocked in a 42°C water bath for 90 seconds. After adding 900ul of non-resistant 2YT culture medium, 37°C, 200rpm shaking culture was revived for 60 minutes. Then about 50ul of bacterial liquid was taken and applied to LB solid culture medium (containing Amp resistance). Incubate overnight at 37°C. On the second day, pick a single clone and inoculate it into 1000 mL of culture medium and incubate at 37°C, 250 rpm until OD 600 When the expression level reaches about 0.8, 1 mM IPTG is added to induce expression for 18 h. Then, the cells are collected by centrifugation in a refrigerated centrifuge.

[0095] 2. Purification: Resuspend the cells with ultrasonic disruption buffer, then ultrasonically disrupt and lyse the cells (240W, 60min). After disruption, centrifuge with a refrigerated centrifuge (16000rpm for 20min) to remove the precipitate, take the supernatant to a new centrifuge tube, centrifuge again at 16000rpm for 40min, and retain the supernatant. Connect the AKTA pipeline, set the flow rate to 8mL / min, rinse with 0.5M NaOH for 2h; then rinse with ddH2O for 2h. Install the purification column, set the flow rate to 8mL / min, rinse with ddH2O for 60mL, then rinse with 0.1M NaOH for 80mL; then rinse with ddH2O for 80mL; finally rinse with 0.01M PBS for 50mL before loading, then load the supernatant after centrifugation, flow rate 4mL / min; wash with 8mM imidazole, and then elute with different concentrations of imidazole linear gradient. Collect the eluate and dialyze into PBS overnight. If the target protein with the tag needs to be removed, an appropriate amount of enterokinase with a His tag can be added, incubated at 4°C for 16 hours, and the flow-through is collected, which is the target collagen COL17A3a, COL17 A3b, COL17A3c, COL17A3d, COL17A3e with the tag removed. The electrophoresis of COL17A3a fusion protein with Trx-his-thrombin-S tag is shown in the figure below. Figure 1 As shown; the theoretical molecular weight of the tagged COL17A3a is 35.4KD, and the apparent molecular weight by SDS-PAGE is approximately 43KD.

[0096] The electrophoresis of COL17A3b fusion protein with Trx-his-thrombin-S tag is shown in Figure 2 As shown; the theoretical molecular weight of the tagged COL17A3b is 34.3KD, and the apparent molecular weight by SDS-PAGE is about 40KD.

[0097] The electrophoresis of COL17A3c fusion protein with Trx-his-thrombin-S tag is shown in Figure 3 As shown; the theoretical molecular weight of the tagged COL17A3c is 33.1KD, and the apparent molecular weight by SDS-PAGE is about 35KD.

[0098] The electrophoresis of COL17A3d fusion protein with Trx-his-thrombin-S tag is shown in Figure 4 As shown; the theoretical molecular weight of the tagged COL17A3d is 19.7KD, and the apparent molecular weight by SDS-PAGE is about 22KD.

[0099] The electrophoresis of COL17A3e fusion protein with Trx-his-thrombin-S tag is shown in Figure 5 The theoretical molecular weight of tagged COL17A3e is 19.7KD, and the apparent molecular weight by SDS-PAGE is about 22KD.

[0100] Example 2: Determination of adhesion activity of COL17A3a, COL17 A3b, COL17 A3c, COL17 A3d, COL17 A3e

[0101] The concentration of the collagen to be tested was detected by ultraviolet absorption method. The specific determination method was as follows: the ultraviolet absorption of the sample at 215nM and 225nM was measured respectively, and then the concentration of the collagen was calculated using the empirical formula C (mg / mL) = 0.144*(A215-A225), A215<1.5.

[0102] Dilute collagen with PBS to a concentration of 0.3 mg / mL; add 100uL to each well of a 96-well ELISA plate and coat overnight at 4°C. Remove the coating solution with a pipette the next day, wash the ELISA plate twice with PBS, then add 200uL of 2% BSA to each well. Incubate at 37°C for 1 hour; then remove the coating solution, wash the ELISA plate twice with PBS, and add 10 5HACAT cells (DMEM + 10% FBS) were added and incubated at 37°C for 2 h. Then the cell culture medium was removed and each well was washed 3 times with PBS. Finally, 100ul of 10% CCK8 detection reagent (Biyuntian, C0042) was added to each well. After incubation at 37°C in the dark for 1 h-3 h, the OD450 value was read. The strength of the OD450 signal can directly reflect the number of adherent cells.

[0103] Results Figure 6 COL17 A3a, COL17 A3b, COL17 A3c, COL17 A3d, and COL17 A3e all exhibited good cell adhesion activity.

[0104] Example 3: COL17A3a, COL17 A3b, COL17 A3c, COL17 A3d, COL17 A3e proliferation activity assay

[0105] HACAT cells were revived and cultured with DMEM + 10% FBS medium, and passaged every 3-4 days. When the cell viability reached above 95%, collagen proliferation activity was detected.

[0106] HACAT cells were digested with trypsin, resuspended in serum-free medium after digestion, and the cells were gently blown off and counted. The cell density was adjusted to 1E5 cells / mL and added to a 96-well cell culture plate at 100uL / well;

[0107] Then dilute the collagen to be tested with serum-free DMEM to a concentration of 0.5 mg / mL, and add 100uL / well to a 96-well cell culture plate. Incubate in a 37°C, 5% CO2 incubator for 72 hours. Then remove the culture supernatant, add serum-free DMEM medium containing 10% CCK8 to each well, and continue incubation for 1-3 hours. Read the OD450 value. The signal intensity of OD450 can directly reflect the proliferation of cells.

[0108] Results Figure 7 COL17 A3a, COL17 A3b, COL17 A3c, COL17 A3d, and COL17 A3e all showed good cell proliferation activity.

Claims

1. A recombinant human type XVII collagen, characterized in that: The recombinant human type XVII collagen is any one of COL17A3a, COL17A3b, COL17A3c, COL17A3d or COL17A3e.

2. The recombinant human type XVII collagen according to claim 1, characterized in that: The amino acid sequence of COL17A3a is as shown in SEQ ID NO.2, or an amino acid sequence modified by amino acid substitution, insertion, replacement, addition and deletion to a certain extent on the basis of SEQ ID NO.2, or an amino acid sequence having greater than 80% homology with the amino acid sequence of SEQ ID NO.2; The amino acid sequence of COL17A 3b is as shown in SEQ ID NO.3, or an amino acid sequence modified by amino acid substitution, insertion, replacement, addition and deletion to a certain extent on the basis of SEQ ID NO.3, or an amino acid sequence having greater than 80% homology with the amino acid sequence of SEQ ID NO.3; 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, replacement, addition and deletion to a certain extent on the basis of SEQ ID NO.4, or an amino acid sequence having greater than 80% homology with the amino acid sequence of SEQ ID NO.4; 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, replacement, addition and deletion to a certain extent on the basis of SEQ ID NO.5, or an amino acid sequence having greater than 80% homology with the amino acid sequence of SEQ ID NO.5; The amino acid sequence of COL17A3e is as shown in SEQ ID NO.6, or an amino acid sequence modified by a certain degree of amino acid substitution, insertion, replacement, addition and deletion based on SEQ ID NO.6, or an amino acid sequence having greater than 80% homology with the amino acid sequence of SEQ ID NO.

6.

3. A recombinant human type XVII collagen according to any one of claims 1-2, characterized in that: A protein tag is added to the N-terminus of the collagen, preferably a Trx-his-thrombin-S tag is added to the N-terminus of the collagen.

4. A method for preparing recombinant human type XVII collagen according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) The amino acid sequence of XVII collagen was retrieved from the protein database NCBI, and the sequence number was Q9UMD9.

3. The amino acid sequence was shown in SEQ ID NO.

1. The conserved region in the sequence was searched using the NCIB BLAST tool. The three-dimensional structure of collagen was predicted using the MODELLER and SWISS-MODEL tools. The structure was optimized and the energy was minimized using molecular dynamics simulation. The potential active functional region was predicted using the molecular docking tool: the amino acid sequence shown in SEQ ID NO.2 to 6; 2) The amino acid sequences shown in SEQ ID NO.2 to 6 were cloned into the Escherichia coli expression vector PET32a; the fragments of the five items were constructed into the PET32a plasmid through the BglII and EcoRI restriction sites, and Trx, his, thrombin and S tag tags were connected to the N-terminus of the target fragment, and an enterokinase restriction site was added to construct PET32a-Trx-his-thrombion-S-A3a, PET32a-Trx-his-thrombion-S-A3b, PET32a-Trx-his-thrombion-S-A3c, PET32a-Trx-his-thrombion-S-A3d, and PET32a-Trx-his-thrombion-S-A3e expression plasmids; the expression plasmids were transformed into competent cells BL21 (DE3) by the heat shock method and cultured to obtain bacteria; 3) Resuspend the cells obtained in step 2) with ultrasonic disruption buffer, then ultrasonically disrupt and lyse the cells, remove the precipitate by centrifugation in a refrigerated centrifuge after disruption, take the supernatant into a new centrifuge tube and centrifuge again, and retain the supernatant; connect the AKTA pipeline, set the flow rate to 8 mL / min, rinse with 0.5 M NaOH; then rinse with ddH2O; install the purification column and rinse, then load the supernatant after centrifugation, with a flow rate of 4 min / min; wash with 8 mM imidazole, then elute with different concentrations of imidazole linear gradient, collect the eluate, and dialyze into PBS overnight; If the target protein with the tag needs to be removed, add enterokinase with a His tag, incubate at 4°C for 16 hours, and collect the flow-through, which is the target collagen COL17A3a, COL17A3b, COL17A3c, COL17A3d, COL17A3e with the tag removed.

5. The method for preparing recombinant human type XVII collagen according to claim 4, characterized in that The specific steps of culturing in step 2) are as follows: take BL21 competent cells, dissolve them on ice, add the expression plasmid, mix them well and let them stand on ice, then heat shock them in a water bath, add 2YT medium without resistance, and culture them on a shaker at 37°C and 200rpm for 60 minutes; spread the bacterial solution on LB solid medium containing Amp resistance, and culture them in a 37°C incubator overnight; then pick a single clone colony and inoculate it into 1000mL of culture medium, culture it at 37°C and 250rpm until OD600 reaches 0.8, add 1mM IPTG to induce expression for 18 hours, and then centrifuge it in a refrigerated centrifuge to collect the bacteria.

6. The method for preparing recombinant human type XVII collagen according to claim 3, characterized in that The specific method for flushing the purification column in step 3) is: set the flow rate to 8 mL / min, flush 60 mL with ddH2O, then flush 80 mL with 0.1 M NaOH; flush 80 mL with ddH2O; and finally flush 50 mL with 0.01 M PBS before loading.

7. A product, comprising the collagen described in any one of claims 1-3 or the collagen or composition prepared by any one of the preparation methods of claims 4-6, wherein the product includes a drug, a pharmaceutical composition, a medical device, a biomaterial, a tissue engineering product, a cosmetic or a health product.

8. Use of the product according to claim 7 in the preparation of medicines, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, cosmetics, health care medicines or health care products for treating skin anti-aging and tissue repair.

9. Use of the product according to claim 7 in the preparation of medicines, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, cosmetics, health care medicines or health care products for treating hair loss and graying hair.

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