A recombinant collagen type XVII and a preparation method and application thereof

Abstract

The application discloses a kind of recombinant collagen XVII and its preparation method and application, belong to the field of bioengineering technology.The amino acid sequence of the recombinant collagen XVII is as shown in SEQ ID NO.9.The application further provides the preparation method of the recombinant collagen XVII, by the optimized coding gene is cloned to pET28a (+) vector, and is induced expression in escherichia coli BL21 (DE3), realizes the efficient soluble production of this complex structure protein.In addition, it is made into composite hydrogel by chemical crosslinking with chitosan, polyglutamic acid.The composite hydrogel has excellent biological activity, mechanical strength and controllable degradability, and shows significant repair-promoting effect in vitro and animal model.The application realizes the whole chain innovation from active protein rational design, efficient expression preparation to high-end medical material application.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant type XVII collagen, its preparation method, and its application. Background Technology

[0002] Collagen is one of the most abundant and structurally crucial extracellular matrix proteins in animals, with 28 different types identified to date. Widely distributed in tissues such as skin, bone, and muscle, it not only provides mechanical support for cells and tissues but also participates in regulating numerous biological processes such as cell adhesion, migration, proliferation, and differentiation, playing an indispensable role in maintaining tissue integrity and repairing damage. Due to its excellent biocompatibility, low immunogenicity, and biodegradability, collagen has become a promising core biomaterial in fields such as biomedicine, tissue engineering, cosmetics, and functional foods.

[0003] Among the many types, type XVII collagen (also known as BP180 or COL17) is a unique transmembrane non-fibrous collagen, primarily expressed in the basal layer of the skin. As a key molecule connecting the intracellular keratinocyte backbone to the extracellular matrix, it plays a crucial role in maintaining the strength of the epidermal-dermal junction, regulating the microenvironment of hair follicle stem cells, and promoting skin homeostasis and regeneration. Its function depends on specific triple-helix domains. However, type XVII collagen is present in extremely low concentrations in natural tissues, making large-scale extraction and purification from animal tissues difficult. Currently, commercially available collagen products are mainly derived from pig, bovine, or fish skin, which presents inherent drawbacks such as the risk of pathogen transmission, immune rejection reactions, and significant batch-to-batch quality variations, severely limiting its application in high-end medical fields.

[0004] Genetic engineering technology offers a fundamental solution for obtaining high-purity, low-risk recombinant human collagen. Researchers have attempted to produce recombinant collagen using various expression systems, such as expression in mammalian or insect cells to obtain near-natural post-translational modifications. However, these systems are costly and have low yields. While the Pichia pastoris system can perform eukaryotic post-translational modifications and achieve high-density fermentation, its long culture cycle, complex induction expression process, and unstable expression levels of exogenous proteins increase the difficulty of process control and large-scale production.

[0005] Among various expression systems, the *E. coli* expression system, with its clear genetic background, low culture cost, short fermentation cycle, and high yield, has become the preferred platform for the industrial production of recombinant proteins. Existing technologies have reported successful expression of type I and type III human collagen in BL21(DE3) using the pET28a vector. However, for the structurally complex and functionally important type XVII collagen, a technical solution for achieving efficient soluble heterologous expression using the pET28a-BL21(DE3) combination remains a gap in existing literature and patents. While Ji Tangbing et al.'s paper, "Efficient Extracellular Expression of Recombinant Human Collagen XVII in *E. coli*", disclosed the use of other *E. coli* strains (such as BL21 ΔdacB) and the pRSFDuet-1 vector to achieve extracellular expression of specific fragments of type XVII collagen, this did not cover, nor could it directly deduce, the expression strategy for type XVII collagen in the pET28a-BL21(DE3) system.

[0006] Further analysis revealed that existing recombinant type XVII collagen preparation technologies suffer from issues with sequence design and sequence uniformity. Most recombinant strategies express only a single collagen domain or a few functional fragments, failing to fully integrate the multiple bioactive sites of type XVII collagen. While some technologies attempt to combine multiple fragments, the fragment selection lacks specificity, resulting in functionally limited and inactive products. This affects their binding ability to natural ligands, thereby weakening their inherent key biological functions such as cell adhesion and signal transduction, making it difficult to meet the high-performance requirements for protein bioactivity in applications such as gel materials.

[0007] In end-use applications such as wound dressings, the market demands higher performance from collagen materials. Traditional dressings are mostly passive covering products, primarily functioning as physical barriers, and their ability to actively promote cell adhesion, proliferation, and migration, as well as efficiently guide tissue regeneration, is generally insufficient. Developing a novel composite material with good bioactivity is key to advancing wound care technology. This requires that the recombinant collagen supplied from upstream not only have high yield and purity, but also possess a complete and efficient structure and function. Therefore, how to design recombinant type XVII collagen sequences with both high expressivity and high functionality, and ultimately transform them into high-performance gel materials that meet clinical needs, remains a core challenge across the entire chain from basic research to product development. Summary of the Invention

[0008] In view of the above-mentioned prior art, the purpose of this invention is to provide a recombinant type XVII collagen, its preparation method, and its application. The aim is to utilize genetic engineering technology, using PET28a as a carrier, E. coliBL21(DE3) was used to heterologously express type XVII collagen, which was then purified and cross-linked with polyglutamic acid and chitosan to form a gel. This method aims to overcome the problems of uncertain recombinant type XVII collagen expression systems, limited sequence design, and insufficient application performance in existing technologies.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a recombinant type XVII collagen, wherein the recombinant collagen is obtained by selecting the 15th collagen domain, the 13th collagen domain, the non-collagen 4th collagen domain and the 1st collagen domain from human type XVII collagen, tandemly connecting them, and repeating the process twice.

[0011] The amino acid sequence of the 15th collagen domain is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the 15th collagen domain is shown in SEQ ID NO.2.

[0012] The amino acid sequence of the 13th collagen domain is shown in SEQ ID NO.3; the nucleotide sequence of the gene encoding the 13th collagen domain is shown in SEQ ID NO.4.

[0013] The amino acid sequence of the non-collagen fourth domain is shown in SEQ ID NO.5; the nucleotide sequence of the gene encoding the non-collagen fourth domain is shown in SEQ ID NO.6.

[0014] The amino acid sequence of the first collagen domain is shown in SEQ ID NO.7; the nucleotide sequence of the gene encoding the first collagen domain is shown in SEQ ID NO.8.

[0015] The amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO.9.

[0016] Preferably, the nucleotide sequence of the gene encoding the recombinant type XVII collagen is shown in SEQ ID NO.10.

[0017] In a second aspect, the present invention provides a method for preparing the above-mentioned recombinant type XVII collagen, comprising the following steps:

[0018] (a) The gene sequence encoding the recombinant type XVII collagen gene was cloned into an expression vector to construct a recombinant expression plasmid;

[0019] (b) Transform the recombinant expression plasmid into an Escherichia coli expression host to obtain recombinant engineered bacteria;

[0020] (c) The recombinant engineered bacteria were fermented to induce the expression of the target protein. The expressed protein product was harvested and purified to obtain the purified recombinant type XVII collagen.

[0021] Preferably, in step (a), the expression vector is pET28a(+); in step (b), the Escherichia coli expression host is BL21(DE3) strain.

[0022] Preferably, in step (c), IPTG is used for induction expression at an induction temperature of 25°C for 21 hours.

[0023] In a third aspect, the present invention provides the use of the above-mentioned recombinant type XVII collagen in the following (1) or (2):

[0024] (1) Prepare products that promote cell adhesion, proliferation, or migration;

[0025] (2) Prepare products that guide tissue regeneration and promote skin wound healing.

[0026] The product uses recombinant type XVII collagen as its active ingredient.

[0027] Preferably, the product may also contain a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient.

[0028] Preferably, the product is a composite hydrogel prepared from recombinant type XVII collagen, chitosan and polyglutamic acid.

[0029] In vitro cell experiments showed that the pure protein group (COL group) directly supplemented with recombinant type XVII collagen in the early stage of culture (24-48 hours) exhibited excellent cell proliferation and migration-promoting activities. Particularly at 24 hours, the COL group showed a superior proliferation-promoting effect compared to the composite hydrogel extract group (Col / CS / y-PGA group); by 48 hours, the Col / CS / y-PGA group surpassed it and maintained a higher level of proliferation promotion. This dynamic change indicates that: firstly, the free recombinant protein can be rapidly utilized by cells, directly demonstrating that the recombinant type XVII collagen prepared in this invention possesses strong and complete biological activity, which is the core basis of its function. Secondly, although the release of the composite hydrogel is slightly slower in the initial stage, it provides more sustained activity support, demonstrating its advantages as a sustained-release carrier.

[0030] It is important to emphasize that the initial design intent of the composite hydrogel was not to surpass pure proteins in short-term in vitro experiments, but rather to address a key bottleneck in clinical applications. Pure protein solutions are easily diluted and rapidly degraded in vivo, making it difficult to maintain an effective concentration and provide physical support at the wound site. In contrast, the Col / CS / γ-PGA composite hydrogel of this invention combines multiple functions, including long-lasting sustained release, mechanical support, and maintenance of a moist environment, providing a superior solution for actively promoting tissue regeneration.

[0031] The preparation method of the composite hydrogel includes the following steps:

[0032] (1) Weigh out chitosan, dissolve it in lactic acid solution, and prepare chitosan solution;

[0033] (2) Add polyglutamic acid aqueous solution and recombinant type XVII collagen to the above chitosan solution, stir evenly to form a mixed system;

[0034] (3) Add a crosslinking agent to the mixture and react and crosslink at a low temperature of 4°C to form a composite hydrogel.

[0035] The beneficial effects of this invention are:

[0036] First, at the protein design level, this invention uses bioinformatics analysis to specifically select the 15th collagen domain, the 13th collagen domain, the non-collagen 4th domain, and the 1st collagen domain from human type XVII collagen. These four functional fragments are tandemly linked and repeated twice to obtain the recombinant sequence of human type XVII collagen (shown in SEQ ID NO. 9). The repetition and combination of domains enhances the stability and functional abundance of the structure, effectively overcoming the defects of single sequence and incomplete active sites in the prior art, and maximizing the maintenance of its native conformation and biological activity.

[0037] Secondly, regarding efficient preparation, a method is provided that utilizes the pET28a(+) support with... E. coli A method for heterologous expression of recombinant type XVII collagen using a combination of BL21(DE3) strains is disclosed and verified for the first time. This invention clearly discloses and verifies the applicability of this expression system to complex type XVII collagen, achieving efficient and soluble expression of type XVII collagen, and filling the gap in existing technologies regarding pET28a(+) vectors and... E. coli The BL21(DE3) strain combination system fills the gap in heterologous expression of type XVII collagen, laying a reliable foundation for industrial production.

[0038] Third, at the high-performance application level, a novel composite hydrogel material and its preparation method are provided, using the aforementioned highly active recombinant type XVII collagen as the key functional component. The purified recombinant type XVII collagen is blended with polyglutamic acid (γ-PGA) and chitosan (CS). Under low-temperature conditions, EDC / NHS chemical cross-linking technology is used to stably bind the active sites of the collagen to the polymer network through amide bonds and electrostatic interactions. This hydrogel material significantly promotes cell adhesion, proliferation, and migration, and exhibits excellent effects in accelerating skin wound healing in animal models, thus achieving a complete chain of innovation from active protein design and efficient expression preparation to high-end medical material applications. Attached Figure Description

[0039] Figure 1 Map of recombinant expression plasmid vectors.

[0040] Figure 2 Electrophoresis image for PCR validation of the recombinant expression plasmid. M: Marker; 1: Detection band.

[0041] Figure 3 This is a verification diagram showing the induced expression of recombinant type XVII collagen at the shake-flask level; in which... Figure 3 In the image, A represents the SDS-PAGE analysis diagram, M represents the marker, 1 represents the supernatant of recombinant lysed cells, and 2 represents the precipitate of recombinant lysed cells. Figure 3 In the diagram, B represents the Western Blot identification image, 1 represents the supernatant of the recombinant cells, and M represents the marker.

[0042] Figure 4 This is a validation diagram of induced expression in a 5 L fermenter; where Figure 4 Growth curve and induced expression process of recombinant strain A; Figure 4 B represents protein SDS-PAGE at different culture times: 1: 3h; 2: 6h; 3: 9h; 4: 12h; 5: 15h; 6: 18h; 7: 21h.

[0043] Figure 5 SDS-PAGE images of recombinant protein purified by nickel column, anion exchange column, and cation exchange column. 1: AKTA Ni column 500mM Elution; 2: Q column puncture; 3: SP column puncture; 4: SP Elution 0.1M; 5: SP Elution 0.3M; 6: SP Elution 0.5M; 7: SP Elution 0.7M; 8: SP Elution 1M.

[0044] Figure 6 Macroscopic morphological image of the Col / CS / y-PGA composite hydrogel.

[0045] Figure 7 The effect of different samples on L929 cell proliferation (MTT assay); among them Figure 7 Figure A shows the cell survival rate statistics after 24 hours of treatment; Figure 7 Figure B shows the cell survival rate statistics after 48 hours of treatment.

[0046] Figure 8 The results are from a cell scratch assay; among which Figure 8 Image A shows the staining of the scratched area at 0 hours, 24 hours, and 48 hours after the scratching. Figure 8 In the middle, B is a quantitative statistical bar chart of cell scratch healing rate.

[0047] Figure 9 The results are from a cell adhesion assay; among which... Figure 9 In the middle, A is the coloring diagram; Figure 9 B in the figure represents a quantitative statistical bar chart of adhesion rate.

[0048] Figure 10 Results of wound healing experiments in animals; among which Figure 10 Image A shows a comparison of wound photographs taken on days 3, 7, and 14 after skin defect surgery in mice from each group. Figure 10 Figure B shows the quantitative statistical curves of the wound healing rate of each group of mice over time. Detailed Implementation

[0049] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0050] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0051] Example 1: Sequence design and synthesis of recombinant type XVII collagen

[0052] 1. Sequence design:

[0053] Four amino acid functional fragments were selected from human type XVII collagen using bioinformatics and protein structure prediction tools (NCBI, UniProt, AlphaFold3, Pymol). These fragments contain three collagen domains and one non-collagen domain: the 15th collagen domain, the 13th collagen domain, the 4th non-collagen domain, and the 1st collagen domain. The four functional fragments were tandemly repeated twice to form the final recombinant sequence (SEQ ID NO. 9). The aim was to maximize the preservation of its native conformation and biological activity, while enhancing its stability and functional abundance through repetition.

[0054] 2. Gene synthesis and cloning:

[0055] The designed amino acid sequence was optimized using E. coli preferred codons, and the entire gene was synthesized by Nanjing GenScript Biotech Co., Ltd. The synthesized gene sequence is shown in SEQ ID NO.10. Nde I and Xho I restriction enzyme sites were introduced at both ends of the synthesized gene. The target gene fragment was cloned into the corresponding sites of the expression vector pET28a(+) through a double enzyme digestion reaction to construct the recombinant expression plasmid. The recombinant expression plasmid vector map is shown below. Figure 1 As shown.

[0056] Example 2: Construction and Identification of Engineered Bacteria

[0057] The recombinant expression plasmid prepared in Example 1 was transformed into E. coli competent cells BL21 (DE3). The specific procedure was as follows:

[0058] (1) Take out the competent Escherichia coli cells BL21(DE3) from the -80 °C refrigerator and place them on ice. When they are half thawed, take 2 μL of the recombinant expression plasmid prepared in Example 1 and add it to the competent Escherichia coli cells BL21(DE3) in a clean bench.

[0059] (2) Place the mixture on ice for 5 minutes, then heat shock it in a 42°C water bath for 90 seconds, and then place it on ice for 5 minutes.

[0060] (3) Add 600 μL of antibiotic-free LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride) to the clean bench and revive for 60 min at 37°C and 220 rpm.

[0061] (4) Take 100 μL of the resuscitated bacterial solution and spread it evenly on an LB plate containing kanamycin (50 mg / ml). Place the plate in an incubator at 37°C and incubate overnight until uniform colonies grow. Then, perform PCR verification.

[0062] PCR verification using universal primers for the vector

[0063] T7: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO. 11);

[0064] T7t: 5'-GCTAGTTATTGCTCAGCGG-3' (SEQ ID NO. 12).

[0065] The PCR conditions were set as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s; 51 °C annealing for 30 s; 72 °C extension for 2 min; 72 °C final extension for 10 min; a total of 30 cycles of denaturation, annealing and extension.

[0066] PCR verification confirmed that the recombinant expression plasmid was successfully inserted into the expression vector pET28a(+), and positive clones produced a band of approximately 1200 bp. Figure 2 ).

[0067] Example 3: Expression of recombinant type XVII collagen

[0068] 1. Shake-flask level expression of recombinant type XVII collagen

[0069] Pick the confirmed single colonies from the plate and transfer them to 10 ml of LB liquid medium (50 μg / m² kanamycin). Incubate at 37°C on a shaker at 220 rpm for approximately 12 hours. Then, transfer the culture to 500 mL of medium and incubate at 37°C on 220 rpm for 3 hours until OD reaches 50%. 600 At 0.6-0.8, add 50 μL of IPTG (isopropyl thio-β-D-galactopyranoside, final concentration 0.1 mM), and induce for 20 h at 25°C and 180 rpm. Centrifuge at 4°C and 8000 rpm for 5 min, collect the cells, sonicate to disrupt, and centrifuge at 12000 rpm to separate the cell lysis supernatant and precipitate. Sample the cell supernatant and precipitate separately, mix with 5X loading buffer, boil in a metal bath at 95°C for 5 min, and analyze by SDS-PAGE and WB.

[0070] SDS-PAGE analysis showed soluble expression, and the molecular weight was consistent with the SDS-PAGE image, approximately 35 kDa, indicating successful expression of recombinant type XVII collagen. Figure 3 ).

[0071] 2. Scale-up and high-yield expression of recombinant type XVII collagen in a 5 L fermenter

[0072] The recombinant engineered bacteria were inoculated into 50 mL of LB primary medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. The culture was then transferred to 300 mL of secondary seed medium (1% peptone, 1% yeast extract, 0.4% sodium chloride, 0.25% potassium dihydrogen phosphate, and 0.133% dipotassium hydrogen phosphate) and cultured at 37°C and 220 rpm for approximately 5 hours. The resulting seed culture was then transferred to a 5 L fermenter containing 3 L of fermentation medium (1.6% peptone, 2.3% yeast extract, 0.4% sodium chloride, 0.25% potassium dihydrogen phosphate, 0.133% dipotassium hydrogen phosphate, 0.07% magnesium sulfate heptahydrate, and 0.25% glucose). Initial fermentation parameters were set as follows: temperature 37°C, rotation speed 350 rpm, aeration 5 N / min, pH 7, and DO ≥ 30%. Once dissolved oxygen levels rise to 80%, begin feeding (feeding medium formula: peptone 1.6%, yeast extract 2.3%, sodium chloride 0.4%, potassium dihydrogen phosphate 0.25%, dipotassium hydrogen phosphate 0.133%, magnesium sulfate heptahydrate 3.25%, glycerol 50%). Dissolved oxygen is controlled by adjusting the rotation speed and feeding rate to maintain it at 30%. Wait for OD... 600 After 10 hours, 1 mM IPTG was added, the temperature was maintained at 37°C, dissolved oxygen was controlled at around 30%, and the induction time was 21 hours. Samples were taken periodically after the induction period began, and the fermentation broth was analyzed by SDS-PAGE.

[0073] The test results showed that under the induction conditions of 25°C, a final IPTG concentration of 1 mmol / L, and after 21 hours of induction and a culture time of 25 hours, the bacterial density OD... 600 The value reached 33.9, and its growth was as follows: Figure 4 As shown in the figure. Bacterial cells at different culture stages were collected, the supernatant was collected after lysis, purified, and the protein concentration was determined by the BCA method. The final yield of recombinant type XVII collagen in a 5 L fermenter reached 2.5 g / L.

[0074] Example 4: Purification of recombinant type XVII collagen

[0075] Based on Example 3, the expressed recombinant type XVII collagen was purified, and the specific procedures are as follows:

[0076] 1. Nickel column purification of recombinant type XVII collagen

[0077] 1) Wash NiNTABeads with 5 column volumes of ddH2O; 2) Add 5 column volumes of LysisBuffer to the column and equilibrate; 3) Add the supernatant filtered through a 0.22 μm filter membrane to the equilibrated gravity column, retaining the sample for at least 2 minutes to ensure sufficient contact between the target protein and the medium, thus improving the recovery rate. Collect the eluent; 4) Wash with 10 column volumes of WashBuffer to remove non-specifically adsorbed proteins, and collect the wash buffer; 5) Elute the target protein with 5 column volumes of ElutionBuffer, and collect the eluent; 6) Equilibrate the packing material with 5 column volumes of deionized water. Store the gravity column in an equal volume of 20% ethanol at 4°C to prevent bacterial contamination of the packing material. Mix the eluent with 5X loading buffer, boil in a metal bath at 95°C for 5 minutes, and determine purity using SDS-PAGE.

[0078] 2. Purification using cation and anion exchange columns:

[0079] The target protein was dialyzed into buffer A (20 mM Tris-HCl, pH 8.0) using a dialysis bag (8000-14000 Da). The flow-through solution was collected using the AKTA system and passed through an anion exchange column (QPurose6FastFlow). The retained solution was then loaded onto a cation exchange column (SPPurose6FastFlow) to collect the high-purity target protein fraction.

[0080] After the above purification process, as follows: Figure 5 As shown, SDS-PAGE electrophoresis revealed a single band, and grayscale analysis indicated a protein purity higher than 90%. The purified protein solution was dialyzed and then lyophilized to obtain a white flocculent powder, which was stored at -20°C for later use.

[0081] Example 5: Preparation of recombinant type XVII collagen-chitosan-polyglutamic acid composite hydrogel (Col / CS / y-PGA)

[0082] 1. Weigh a certain amount of chitosan (CS), dissolve it in a 0.6% (w / v) lactic acid solution, stir magnetically until completely dissolved, and prepare a 3% (w / v) CS solution.

[0083] 2. Mix the above CS solution with an equal volume of 3% (w / v) polyglutamic acid (γ-PGA) aqueous solution.

[0084] 3. Continue to add the freeze-dried recombinant type XVII collagen powder prepared in Example 4, and slowly stir to disperse the protein evenly, so that the final concentration of recombinant type XVII collagen in the solution is 10 mg / mL.

[0085] 4. Add crosslinking agents EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). The molar ratio of EDC to NHS is 1:1. The amount of crosslinking agent added is 2% of the total mass of the mixture. Let it stand at 4°C for 12 hours to crosslink into a gel.

[0086] After the reaction is complete, an opaque, elastic three-dimensional network hydrogel is formed. Figure 6 The gel undergoes an amide bond reaction catalyzed by EDC / NHS, which covalently crosslinks the amino and carboxyl groups of collagen, CS, and γ-PGA, while electrostatic interactions also exist among the three components.

[0087] Comparative Example 1: Preparation of chitosan-polyglutamic acid composite hydrogel (CS / γ-PGA)

[0088] Compared to Example 5, no lyophilized recombinant type XVII collagen powder was added during the preparation of the composite hydrogel in Comparative Example 1. The specific preparation method is as follows:

[0089] 1. Weigh a certain amount of chitosan (CS), dissolve it in a 0.6% (w / v) lactic acid solution, stir magnetically until completely dissolved, and prepare a 3% (w / v) CS solution.

[0090] 2. Mix the above CS solution with an equal volume of 3% (w / v) polyglutamic acid (γ-PGA) aqueous solution.

[0091] 3. Add crosslinking agents EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). The molar ratio of EDC to NHS is 1:1, and the amount of crosslinking agent added is 2% of the total mass of the mixture. Let it stand at 4°C for 12 hours to crosslink into a gel.

[0092] Experimental Example 1: Evaluation of the bioactivity of composite hydrogels

[0093] The preparation method of the hydrogel extract used in this experiment is as follows: The recombinant type XVII collagen-chitosan-polyglutamic acid composite hydrogel (Col / CS / y-PGA) prepared in Example 6 and the chitosan-polyglutamic acid composite hydrogel (CS / y-PGA) prepared in Comparative Example 1 were placed in serum-free DMEM medium (DMEM medium + 1% penicillin-streptomycin), with 10 mL of serum-free DMEM medium added per gram of hydrogel. The extract was incubated at 37°C and 5% CO2 for 24 hours to obtain the extract. Subsequently, the extract was filtered through a 0.22 μm sterile filter membrane, aliquoted, and stored at 4°C for later use, and used within 48 hours.

[0094] 1. Cell proliferation experiment

[0095] The specific experimental procedures are as follows: (1) Take out L929 cells with a cell confluence of about 80%, discard the complete culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin) in the T25 culture flask, and slowly wash the cells with 2 mL of PBS from the edge of the culture dish to remove metabolic waste and residual culture medium. Add 1 mL of trypsin to the culture flask, tilt the culture flask to allow the trypsin to fully wet the cell surface, and place it in a CO2 incubator at 37°C for 3 min for digestion.

[0096] (2) Observe the cell morphology and gaps under a microscope. When the cell morphology becomes rounder and the gaps become larger, add 2 mL of basal culture medium (DMEM culture medium) to stop digestion and pipette the adherent cells into the suspension.

[0097] (3) Centrifuge the cell suspension at 1000 rpm for 5 min, seed the cells into 96-well plates, add 100 μL of complete culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin) to each well, and incubate overnight at 37°C in a CO2 incubator.

[0098] (4) Discard the culture medium, wash three times with PBS, and set up blank group, control group and experimental group (CS / y-PGA group, Col / CS / y-PGA group and COL group). The treatment of each group is as follows:

[0099] Control group: Add complete culture medium, without cells.

[0100] Control group: Cells were cultured normally with complete culture medium.

[0101] CS / y-PGA group: Add CS / y-PGA extract + 10% fetal bovine serum and culture cells normally.

[0102] Col / CS / y-PGA group: Add Col / CS / y-PGA extract + 10% fetal bovine serum, and culture cells normally.

[0103] Group COL: The lyophilized recombinant type XVII collagen powder prepared in Example 4 was dissolved in complete culture medium (DMEM medium + 10% fetal bovine serum + 1% penicillin-streptomycin) to prepare a series of solutions, so that the final concentrations of recombinant type XVII collagen in the cell culture system were 0.06 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL and 4 mg / mL, respectively, and cells were cultured normally at different concentrations.

[0104] Each group has 5 duplicate holes.

[0105] (5) Add 10 μL of MTT solution to each well of the control group and the experimental group. Incubate in the dark for 4 hours.

[0106] (6) Add 150 μL of DMSO to each well in both the control and experimental groups. Shake on a shaker until the blue-purple crystals are completely dissolved, and then measure the OD value of each group at 490 nm using a microplate reader.

[0107] The formula for calculating cell viability is as follows:

[0108] Cell viability (%) = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%

[0109] Figure 7 The results showed that after 24 hours of culture, the cell proliferation rate in the CS / y-PGA group did not change significantly compared with the control group. The COL group showed a concentration-dependent effect, with the 1 mg / mL concentration showing the most significant proliferative effect and a cell viability rate as high as 129%; while the 4 mg / mL concentration group exhibited cytotoxicity, with a significantly reduced cell viability rate, presumably because this recombinant protein is a basic protein, and excessively high concentrations inhibit cell growth. The Col / CS / y-PGA group demonstrated significant proliferative activity.

[0110] After 48 hours of culture, compared with the control group: in the COL group, the 1 mg / mL concentration group showed a further enhanced proliferative effect, with a cell viability of 144%. The proliferative effect of the Col / CS / y-PGA group was also significant, with a cell viability of 145%. The proliferative effects of both groups (the optimal concentration of the COL group and the Col / CS / y-PGA group) were significantly better than those of the control group and the CS / y-PGA group, and there was no statistically significant difference between the two groups (the optimal concentration of the COL group and the Col / CS / y-PGA group).

[0111] 2. Cell scratch migration assay:

[0112] The ability of recombinant collagen to promote L929 cell migration was detected by cell scratch assay.

[0113] (1) Cell plating and scratches:

[0114] Draw three evenly spaced horizontal lines on the back of a 6-well plate using a marker, with each line spaced 0.5–1 cm apart. Inoculate each well with 6 × 10⁶ cells / well. 5 Add 2 mL of complete culture medium to each L929 cell and incubate at 37°C with 5% CO2 for 24 hours to allow the cells to form a dense monolayer. Using the tip of a 200 μL sterile pipette, make a straight, even scratch along a horizontal line perpendicular to the back of the plate in one stroke. Discard the old culture medium and gently wash the plate three times with sterile PBS to remove the scratched cell debris.

[0115] (2) Experimental grouping and treatment: After washing, 2 mL of different test samples were added to each of the 6-well plates. The groups are as follows:

[0116] Control group: serum-free DMEM medium.

[0117] CS / y-PGA group: CS / y-PGA hydrogel extract.

[0118] Col / CS / y-PGA group: Col / CS / y-PGA composite hydrogel extract.

[0119] Group COL: The lyophilized recombinant type XVII collagen powder prepared in Example 4 was dissolved in serum-free DMEM medium (DMEM medium + 1% penicillin-streptomycin) to prepare a series of solutions, so that the final concentrations of recombinant type XVII collagen in the cell culture system were (0.25 mg / ml, 0.5 mg / ml and 1 mg / ml).

[0120] Return the culture plate to the incubator. Immediately after scratching (0 h), and at 24 h and 48 h after incubation, take three field-of-view photographs along each scratch at the reference line position using an inverted microscope. Measure the scratch area at each time point using ImageJ software. Calculate the scratch healing rate using the following formula:

[0121] Scratch healing rate = (Initial scratch area - Scratch area at a certain moment) / Initial scratch area

[0122] (3) Results: The results of the scratch test are as follows Figure 8 As shown in the figure, the migration-promoting effect of the CS / y-PGA group was not significantly different from that of the control group. Compared with the control group, both the Col / CS / y-PGA group and the COL group significantly promoted the migration of L929 cells. After 24 h of culture, the scratch healing rate of the Col / CS / y-PGA group was 75%; the healing rate of the 1 mg / ml group in the COL group reached 79%. After 48 h of culture, the healing rate of the Col / CS / y-PGA group increased to 83%; the healing rate of the 1 mg / ml group in the COL group further increased to 87%.

[0123] 3. Cell adhesion experiment

[0124] (1) Sample coating: Add 100 μL of each of the following samples to the wells of a 24-well plate, ensuring the liquid covers the bottom of the well, and coat overnight at 4°C:

[0125] CS / y-PGA group: CS / y-PGA hydrogel extract.

[0126] Col / CS / y-PGA group: Col / CS / y-PGA composite hydrogel extract.

[0127] Group COL: The lyophilized recombinant type XVII collagen powder prepared in Example 4 was dissolved in serum-free DMEM medium (DMEM medium + 1% penicillin-streptomycin) to prepare a series of solutions, so that the final concentrations of recombinant type XVII collagen in the cell culture system were (0.25 mg / ml, 0.5 mg / ml and 1 mg / ml).

[0128] Positive control group (Fibronectin): Human fibronectin powder was dissolved in serum-free DMEM medium to prepare a 100 μg / ml solution.

[0129] Negative control group (Control): Serum-free DMEM medium.

[0130] (2) Blocking and cell seeding: Discard the coating solution, add 1 mL of 1% BSA solution to each well, and block at room temperature for 1 hour. Wash each well 3 times with PBS. After digestion and counting, seed the L929 cells at 6 × 10⁶ cells per well. 5 Cells were seeded at a density of 500 μL of cell suspension into pretreated wells, with three replicates per group. The culture plates were then incubated at 37°C in a 5% CO2 incubator for 3 hours.

[0131] (3) Fixation, staining, and quantification: After culture, carefully aspirate the culture medium and gently wash the well plate three times with pre-warmed PBS to remove unattached cells. Add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 15 minutes. After washing with PBS, add 1 mL of 0.1% crystal violet staining solution to each well and stain for 20 minutes. Gently rinse the well plate with running water until no free dye remains, and photograph cell adhesion under a microscope. After photographing, add 1 mL of 33% acetic acid solution to each well and shake on a shaker for 10 minutes to completely dissolve the crystal violet. Transfer 200 μL of the solution to a 96-well plate and measure the absorbance (OD) value at 595 nm using a microplate reader.

[0132] The results of the cell adhesion experiment are as follows Figure 9 As shown. Compared with the negative control group, all experimental groups coated with collagen samples showed significant cell adhesion activity, normalized to 100% of the OD value of the negative control group. The bacterial adhesion rate of the positive control group was 152% of that of the negative control group, the cell adhesion rate of the Col / CS / y-PGA group was 148% of that of the negative control group, and the cell adhesion rate of the 1 mg / ml COL group was 149% of that of the negative control group. The adhesion activity of the CS / y-PGA group was similar to that of the negative control group, but significantly lower than that of the collagen-containing groups.

[0133] Experiment Example 2: Animal Wound Healing Experiment

[0134] Healthy KM mice (4 weeks old, about 20g) were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, their back hair was shaved, and their skin was disinfected routinely. A 6mm diameter wound area was punched using a sterile punch. The treated mice were randomly divided into four groups: (1) Control group: 0.9% saline solution was applied topically; (2) CS / y-PGA group: the composite hydrogel prepared in Comparative Example 1 was used for coverage; (3) Col / CS / y-PGA group: the composite hydrogel prepared in Example 6 was used for coverage; (4) Col group: the lyophilized recombinant type XVII collagen powder prepared in Example 4 was dissolved in PBS solution to prepare a 1 mg / mL recombinant collagen solution, and the 1 mg / mL recombinant collagen solution was applied topically.

[0135] Medication was administered every other day for each treatment. The wound was photographed on days 3, 7, and 14 after treatment, and the wound area was measured using ImageJ to calculate the wound healing rate.

[0136] Wound healing rate = ((initial wound area - wound area on a certain day) / initial wound area) 100%

[0137] The test results showed that compared with the Control group and the CS / y-PGA group, the wound healing speed of the Col / CS / y-PGA group and the Col group was significantly faster. At day 14, the wound healing rate of both the Col / CS / y-PGA group and the Col group reached over 95%, with significant wound contraction, more complete new epithelial tissue, and milder inflammatory response. Figure 10 This indicates that recombinant type XVII collagen and its composite hydrogel can effectively promote the repair of full-thickness skin defects.

[0138] Experimental Example 3: Functional Comparison and synergistic Effect Verification of Different Recombinant Sequences

[0139] Col15-13-NC4-1×2 group (the group of this invention): (Col15 - Col13 - NC4 - Col1) series units are repeated twice.

[0140] Col15-1×2 group: The tandem unit of collagen domain 15 (Col15) and collagen domain 1 (Col1) is repeated twice.

[0141] Col15-13-1×2 group: The 13th collagen domain (Col13) is inserted between Col15 and Col1, and the tandem unit is repeated twice.

[0142] Col15-NC4-1×2 group: A non-collagenous fourth domain (NC4) is inserted between Col15 and Col1, and the tandem unit is repeated twice.

[0143] Col15-13-NC4-1×1: Contains all four target structural domains, but only concatenates them once (Col15 - Col13 -NC4 - Col1).

[0144] The coding genes of the above sequences were all optimized using E. coli preferred codons and synthesized in their entirety by the company. Each gene was cloned into the pET28a(+) vector and transformed into E. coli BL21(DE3) competent cells to construct the corresponding recombinant engineered bacteria. The expression, fermentation, and purification methods for each engineered bacterium were completely identical to those described in Examples 3 and 4 of this invention.

[0145] After induction in a 5L fermenter, the cells of each recombinant engineered bacteria were collected, broken, and centrifuged. The supernatant (soluble fraction) and precipitate (inclusion body fraction) were taken for SDS-PAGE analysis. The target bands were semi-quantitatively analyzed using grayscale scanning software, and the proportion of soluble expression (soluble protein amount / total protein amount × 100%) was calculated.

[0146] Experimental results show that:

[0147] In the Col15-1×2 group, only 32% of the protein was soluble, with most proteins existing as inclusion bodies. This indicates that the basic scaffold has limited solubility and correct folding ability, which is not conducive to industrial production.

[0148] Col15-13-1×2 group: the proportion of soluble expression increased to 55%.

[0149] Col15-NC4-1×2 group: the proportion of soluble expression increased to 58%.

[0150] (The above results indicate that adding either the Col13 or NC4 domain alone can effectively improve protein solubility, suggesting that they play a positive role in proper folding.)

[0151] Col15-13-NC4-1×1: The proportion of soluble expression was significantly increased to 75%. This demonstrates that the complete combination of the four domains is essential for maintaining the solubility and stability of the protein within the host bacteria.

[0152] The sequence of this invention (Col15-13-NC4-1×2) exhibits a soluble expression rate of 89%, significantly higher than all comparative sequences (p<0.01). This not only demonstrates the significant advantage of this sequence in industrial production (high solubility) but also suggests from a physicochemical perspective that its repetitive modular design may promote the formation of more stable, higher-order conformations closer to the natural triple helix structure.

[0153] In summary, the specific amino acid sequence shown in SEQ ID NO:9 of this invention, through its unique and optimized selection, arrangement, and repeating configuration of structural domains, produces a synergistic effect that cannot be achieved by simply superimposing the components.

[0154] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted.

[0155] Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. A recombinant type XVII collagen, characterized in that, Recombinant type XVII collagen was obtained by selecting the 15th collagen domain, the 13th collagen domain, the non-collagenous 4th domain, and the 1st collagen domain from human type XVII collagen, tandemly, and repeating twice; the amino acid sequence of recombinant type XVII collagen is shown in SEQ ID NO.

9.

2. The recombinant type XVII collagen according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the recombinant type XVII collagen is shown in SEQ ID NO.

10.

3. A method for preparing recombinant type XVII collagen, characterized in that, Includes the following steps: (a) The gene sequence of the recombinant type XVII collagen encoding gene as described in claim 2 is cloned into an expression vector to construct a recombinant expression plasmid; (b) Transform the recombinant expression plasmid into an Escherichia coli expression host to obtain recombinant engineered bacteria; (c) The recombinant engineered bacteria were fermented to induce the expression of the target protein. The expressed protein product was harvested and purified to obtain the purified recombinant type XVII collagen.

4. The preparation method according to claim 3, characterized in that, In step (a), the expression vector is pET28a(+); in step (b), the Escherichia coli expression host is BL21(DE3) strain.

5. The preparation method according to claim 3, characterized in that, In step (c), IPTG was used to induce expression at a temperature of 25°C for 20 hours.

6. The use of the recombinant type XVII collagen according to claim 1 in (1) or (2) below, characterized in that: (1) Prepare products that promote the adhesion, proliferation or migration of L929 cells; (2) Prepare products that guide the regeneration of mouse skin epithelial tissue and promote the healing of mouse epithelial tissue skin wounds.

7. The application according to claim 6, characterized in that, The product uses recombinant type XVII collagen as its active ingredient.

8. The application according to claim 7, characterized in that, The product is a composite hydrogel prepared from recombinant type XVII collagen, chitosan, and polyglutamic acid.

9. The application according to claim 8, characterized in that, The preparation method of the composite hydrogel includes the following steps: (1) Weigh out chitosan, dissolve it in lactic acid solution, and prepare chitosan solution; (2) Add polyglutamic acid aqueous solution and recombinant type XVII collagen to the above chitosan solution, stir evenly to form a mixed system; (3) Add a crosslinking agent to the mixture and react and crosslink at 4°C to form a composite hydrogel.

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