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

Abstract

The application discloses a kind of recombinant XVII type humanized collagen and its preparation method and application, it is related to genetic engineering technical field.The recombinant XVII type humanized collagen includes recombinant collagen single chain;Wherein, the recombinant collagen single chain is by the collagen monomer fragment as shown in SEQ ID NO.1, the number of repetitions is 2~10 times.Amino acid sequence of the application is completely derived from the tailoring and splicing of human source XVII type collagen natural sequence, does not introduce any non-human exogenous amino acid residue or non-natural connecting peptide.The application is accurately knocked out to Pichia pastoris host cell YPS1 gene, the activity of host endogenous protease is inhibited from source, and the degradation of target protein in fermentation process is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant type XVII humanized collagen, its preparation method, and its application. Background Technology

[0002] Type XVII collagen is a unique type II transmembrane protein that, as a core structural component of hemidesmosomes, plays a crucial role in the adhesion and connection between epidermal and epithelial cells and the basement membrane. Competition among epidermal stem cells driven by type XVII collagen is key to delaying or reversing skin aging; epidermal stem cells highly expressing type XVII collagen are firmly anchored to the basement membrane and displace nearby cells with low expression of type XVII collagen through symmetrical division. Furthermore, type XVII collagen plays an irreplaceable role in maintaining hair follicle stem cell activity and promoting wound healing. However, due to its extremely low abundance in human and animal tissues, and its long amino acid chain and large molecular weight, natural extraction of type XVII collagen is extremely difficult, hindering large-scale production. Therefore, recombinant expression of type XVII collagen through genetic engineering has become a research hotspot in this field.

[0003] Currently, the preparation technology of recombinant type XVII collagen has been developed to a certain extent. For example, Chinese patent CN118955691A discloses a recombinant humanized type XVII collagen. Its technical solution is as follows: the collagen includes basic repeating units, with at least one basic repeating unit. When the number is greater than or equal to two, adjacent repeating units are connected by peptide bonds. This recombinant collagen has 100% sequence identity with human collagen, with no risk of rejection or sensitization, and exhibits good cell proliferation-promoting activity, cell adhesion-promoting activity, and tissue regeneration and repair capabilities. This patent uses an E. coli expression system for expression. Although it is low-cost and has a short cycle, it lacks eukaryotic post-translational modification, and intracellular expression requires bacterial lysis, which introduces a large amount of impurity proteins and endotoxins.

[0004] Pichia pastoris expression systems, as eukaryotic microorganisms, possess complete post-translational protein modification capabilities, and recombinant proteins can be secreted extracellularly, avoiding contamination issues caused by cell lysis, thus offering significant advantages in industrial production. However, exogenous recombinant proteins expressed in Pichia pastoris are easily non-specifically cleaved by host-secreted proteases during fermentation, generating numerous degradation fragments. This not only reduces the yield and purity of the target protein but also significantly increases downstream purification costs. Chinese patent CN116375847A discloses a yeast recombinant type XVII humanized collagen and its preparation method. This patent is the first to express the full-length sequence of the 15th helical region of XVII collagen in Pichia pastoris, establishing a complete fermentation and purification process. This patent employs a strategy of modifying the collagen amino acid sequence to reduce degradation. However, the amino acid sequence is the cornerstone of protein physicochemical properties and biological functions. Improving anti-degradation performance by mutating the native sequence alters the protein's native conformation at the molecular level, thereby affecting its normal binding ability to receptors such as integrins, and carries the risk of sacrificing biological activity.

[0005] Therefore, developing a recombinant type XVII collagen with 100% human sequence coverage, precise enrichment of highly active functional domains, and effective resistance to fermentation degradation, as well as its preparation method, has significant technical value and application prospects for meeting the urgent need for high-quality collagen materials in the biomedical field. Summary of the Invention

[0006] Therefore, this invention provides a recombinant type XVII humanized collagen, its preparation method, and its application to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a recombinant type XVII humanized collagen protein comprises collagen single chains; wherein the collagen single chain is composed of collagen monomer fragments as shown in SEQ ID NO.1 repeated 2 to 10 times. As an example, preferably, the collagen single chain contains 2 to 6 collagen monomer fragments; more preferably, repeated 2 to 3 times.

[0008] SEQ ID NO.1: GPQGPKGDKGDPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQGPKGDQGPPGPRGHQGEQGPKGDQGPPPGPRGHQGEQGPKGDQGPPGPRGHQGEQ.

[0009] Furthermore, the recombinant collagen single chain is obtained by cutting and splicing the human type XVII collagen α1 chain; wherein the amino acid sequence of the human type XVII collagen is shown in SEQ ID NO.2.

[0010] SEQ ID NO.2:

[0011] Furthermore, the collagen monomer fragment is obtained by sequentially combining and repeating amino acids at positions 957-968, 1441-1482, 914-932, 914-932, and 914-932 of human type XVII collagen twice.

[0012] Furthermore, the amino acid sequence of the recombinant collagen single chain is shown in SEQ ID NO.3.

[0013] SEQ ID NO.3: GPQGPKGDKGDPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQGPKGDQGPPPGPRGHQGEQGPKGDQGPPPGPRGHQGEQGPKGDQGPPGPRGHQGEQ GPQGPKGDKGDPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQGPKGDQGPPPGPRGHQGEQGPKGDQGPPGPRGHQGEQGPKGDQGPPGPRGHQGEQ.

[0014] Furthermore, the nucleotide sequence encoding the collagen single chain is shown in SEQ ID NO.4.

[0015] SEQ ID NO.4: .

[0016] The amino acid sequence of this invention is entirely derived from the combination of natural human type XVII collagen sequences, without introducing any non-human exogenous amino acid residues. Theoretically, its sequence is completely consistent with the corresponding region in the human body, thus exhibiting extremely low immunogenicity and avoiding the risk of viruses carried by animal-derived collagen and allergic reactions caused by foreign proteins.

[0017] A method for preparing recombinant collagen single chains according to a second aspect of the present invention includes the following steps: Step 1: Design collagen sequences The amino acids at positions 957-968, 1441-1482, 914-932, 914-932, and 914-932 of human type XVII collagen are sequentially combined to obtain collagen monomer fragments. The monomer fragments are repeated 2 to 10 times to obtain recombinant collagen single chains. Step 2, constructing the carrier Based on the recombinant collagen single-chain, the corresponding nucleotide sequence was synthesized to construct a recombinant plasmid; Step 3: Transformation and Cultivation The recombinant plasmid was transferred into host cells, engineered strains were screened, and fermentation culture was performed to induce the expression of the target protein. Step 4: Harvesting and Purification The fermentation broth was collected, the expressed protein product was harvested, and purified to obtain the target collagen.

[0018] Furthermore, in step two, the plasmid is selected for prokaryotic expression, specifically the pET series vector, pGEX series vector, pBAD series vector, or pUC series vector; or the plasmid is selected for eukaryotic expression, specifically the pcDNA series vector, pCMV series vector, pIRES series vector, pEGFP series vector, pYES series vector, or pPIC series vector. As an example, preferred plasmids include pcDNA3.1, pCMV-Tag, pCMV-Sport, pIRES2-EGFP, pEGFP-C1, pYES2, pPICZ, or pPIC3.5.

[0019] Furthermore, in step three, the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell. For example, the prokaryotic cell is *Escherichia coli* or *Bacillus subtilis*; the yeast cell is *Pichia pastoris* or *Saccharomyces cerevisiae*; and the mammalian cell is HEK293 cell or CHO cell.

[0020] The stable type XVII recombinant collagen prepared by the method described above, according to a third aspect of the present invention, is used in the preparation of collagen-containing products.

[0021] Furthermore, the collagen product is a cosmetic, medical device, or drug.

[0022] Optionally, the drug includes wound repair materials, cartilage repair materials, and drug delivery carriers; Optionally, the medical device includes tissue engineering materials, surgical aids, and dental prosthetic materials; Optionally, the cosmetics include skin care products, hair care products, and anti-aging products.

[0023] In one embodiment, the medicine includes, but is not limited to, wound healing materials, such as collagen sponge (to promote wound healing), collagen hemostatic dressing (for rapid hemostasis), and collagen membrane (for postoperative repair). Cartilage repair materials, such as collagen injections (for repairing articular cartilage defects) and collagen-based cartilage grafts; Drug delivery carriers, such as collagen microspheres (encapsulating active ingredients for sustained drug release) and collagen gel injections (for local delivery of anti-inflammatory or anti-tumor drugs).

[0024] In one embodiment, the medical device includes, but is not limited to, tissue engineering materials, such as collagen scaffolds (for cell culture and tissue regeneration), collagen-based composite materials (for bone or soft tissue repair), and collagen filler materials (for filling tissue or cavities). Surgical aids include, but are not limited to, collagen sutures (biodegradable sutures) and collagen hemostatic sponges (for intraoperative hemostasis). Oral restoration materials include, but are not limited to, collagen membranes (alveolar bone filling) and collagen periodontal regeneration materials.

[0025] In one embodiment, cosmetics include skincare products, such as collagen masks (moisturizing, repairing, and anti-aging), collagen serums (improving skin elasticity), and collagen lotions (hydrating and locking in moisture). Hair care products, such as collagen shampoo (to repair damaged hair) and collagen conditioner (to replenish nutrients and make hair smooth). Anti-aging products, such as collagen anti-wrinkle cream (to improve skin firmness) and collagen freeze-dried powder (to promote skin regeneration and repair fine lines).

[0026] A fourth object of the present invention is to provide a collagen product containing any of the above-described recombinant collagen single chains; Optionally, the collagen product may be a pharmaceutical product, a medical device, or a cosmetic product.

[0027] Optionally, the drug includes wound repair materials, cartilage repair materials, and drug delivery carriers; Optionally, the medical device includes tissue engineering materials, surgical aids, and dental prosthetic materials; Optionally, the cosmetics include skin care products, hair care products, and anti-aging products.

[0028] In one embodiment, the medicine includes, but is not limited to, wound healing materials, such as collagen sponge (to promote wound healing), collagen hemostatic dressing (for rapid hemostasis), and collagen membrane (for postoperative repair). Cartilage repair materials, such as collagen injections (for repairing articular cartilage defects) and collagen-based cartilage grafts; Drug delivery carriers, such as collagen microspheres (encapsulating active ingredients for sustained drug release) and collagen gel injections (for local delivery of anti-inflammatory or anti-tumor drugs).

[0029] In one embodiment, the medical device includes, but is not limited to, tissue engineering materials, such as collagen scaffolds (for cell culture and tissue regeneration), collagen-based composite materials (for bone or soft tissue repair), and collagen filler materials (for filling tissue or cavities). Surgical aids include, but are not limited to, collagen sutures (biodegradable sutures) and collagen hemostatic sponges (for intraoperative hemostasis). Oral restoration materials include, but are not limited to, collagen membranes (alveolar bone filling) and collagen periodontal regeneration materials.

[0030] In one embodiment, cosmetics include skincare products, such as collagen masks (moisturizing, repairing, and anti-aging), collagen serums (improving skin elasticity), and collagen lotions (hydrating and locking in moisture). Hair care products, such as collagen shampoo (to repair damaged hair) and collagen conditioner (to replenish nutrients and make hair smooth). Anti-aging products, such as collagen anti-wrinkle cream (to improve skin firmness) and collagen freeze-dried powder (to promote skin regeneration and repair fine lines).

[0031] The present invention has the following advantages: This invention combines five highly hydrophilic and stable key functional domains (positions 957-968, 1441-1482, and 914-932) from the α1 chain of human type XVII collagen, eliminating numerous non-functional or low-activity regions. This results in a recombinant protein containing the core active epitopes of natural type XVII collagen within a compact structure of only 216 amino acids. The resulting recombinant collagen exhibits significant promoting effects on cell proliferation, cell adhesion, and cell migration, demonstrating excellent biological activity.

[0032] This invention precisely knocks out the YPS1 gene in Pichia pastoris host cells, thereby inhibiting the activity of endogenous proteases in the host from the source and significantly reducing the degradation of the target protein during fermentation.

[0033] The amino acid sequence of this invention is entirely derived from the cutting and splicing of the natural sequence of human type XVII collagen, without introducing any non-human exogenous amino acid residues or non-natural linker peptides. The 100% human sequence coverage fundamentally eliminates the risk of animal-derived collagen carrying viruses and greatly reduces the risk of immunogenicity and allergic reactions caused by foreign proteins, providing extremely high safety guarantees for high-end medical aesthetics, regenerative medicine, and clinical treatment.

[0034] The recombinant type XVII collagen prepared by this invention has a typical triple helix structure, consistent with the triple helix conformation of natural collagen. This complete higher-order structure ensures that it can fully exert the biological functions of natural type XVII collagen. Attached Figure Description

[0035] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0036] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0037] Figure 1 This is an expression plasmid map of recombinant type XVII humanized collagen COL17 provided in Example 1 of the present invention.

[0038] Figure 2 This is the Pichia pastoris YPS1 gene knockout method provided in Example 1 of the present invention.

[0039] Figure 3 This is an SDS-PAGE electrophoresis pattern of COL17 fermentation provided in Example 1 of the present invention. Wherein, 1 is a BSA electrophoresis pattern; 2 is an electrophoresis pattern of the supernatant of GS115-COL17 fermentation broth; and 3 is an electrophoresis pattern of the supernatant of GS115-YPS1-COL17 fermentation broth.

[0040] Figure 4 This is an SDS-PAGE electrophoresis pattern of COL17 protein purification provided in Example 1 of the present invention; wherein, M is a protein marker electrophoresis pattern; 1 is a BSA electrophoresis pattern; 2 is a flow-through electrophoresis pattern; and 3-4 are elution electrophoresis patterns.

[0041] Figure 5 The results show the thermal stability of the recombinant type XVII humanized collagen provided in Test Example 1 of this invention; where M is a protein marker electrophoresis image, 1 is a 0h electrophoresis image, 2 is a 1h electrophoresis image, 3 is a 2h electrophoresis image, 4 is a 3h electrophoresis image, and 5 is a 4h electrophoresis image.

[0042] Figure 6The results of the acid-base tolerance test of recombinant type XVII humanized collagen provided in Test Example 1 of this invention are shown below; where M is a protein marker electrophoresis image, 1 is a control electrophoresis image, 2 is a pH 6.0 0h electrophoresis image, 3 is a pH 6.5 0h electrophoresis image, 4 is a pH 7.0 0h electrophoresis image, 5 is a pH 7.5 0h electrophoresis image, 6 is a pH 8.0 0h electrophoresis image, 7 is a pH 6.0 24h electrophoresis image, 8 is a pH 6.5 24h electrophoresis image, 9 is a pH 7.0 24h electrophoresis image, 10 is a pH 7.5 24h electrophoresis image, and 11 is a pH 8.0 24h electrophoresis image.

[0043] Figure 7 The results of cell adhesion activity, cell proliferation activity and cell migration detection of the recombinant collagen provided in Test Example 1 of this invention; Figure 8 The results of the recombinant collagen cytotoxicity test provided in Test Example 1 of this invention; Figure 9 This is a graph showing the evaluation results of diabetic wound healing provided in Test Example 2 of the present invention; Figure 10 The image shows a circular dichroism (CD) chromatogram of COL17 protein provided in Test Example 4 of this invention. Detailed Implementation

[0044] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0046] Yeast extract: Oxoid, LP0021B; Peptone: Oxoid, LP0042B; YNB: Shanghai Yuanye Biotechnology Co., Ltd., S24483; Glycerin: Ruikangda, GY20250113; Biotin: Aladdin, D2317484; Potassium phosphate buffer: Beijing Solarbio Science & Technology Co., Ltd., P1030.

[0047] Seed culture medium YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose); BMGY medium (1% yeast extract + 2% peptone + 1.34% YNB + 1% glycerol + 0.0004% biotin + 100mM potassium phosphate buffer, pH 6.0). BMMY medium (1% yeast extract + 2% peptone + 1.34% YNB + 0.0004% biotin + 100mM potassium phosphate buffer, pH 6.0). Fermentation medium (glycerol 46 g / L, magnesium sulfate heptahydrate 2.02 g / L, calcium sulfate dihydrate 0.46 g / L, potassium sulfate 9.1 g / L, phosphate 11.34 mL / L, potassium hydroxide 2.06 g / L, PTM1 trace element 4 mL / L); fed-batch medium (50% w / v glycerol, with 12 mL PTM1 trace element per liter). Induction medium (100% methanol, with 12 mL of PTM1 trace elements added per liter); PTM1: sterilized by filtration through a 0.22 μm filter membrane and stored at 4°C. After the fermentation medium is sterilized at high temperature, PTM1 is added after the temperature drops to room temperature, and the pH is adjusted to 5.0 with ammonia. The pPIC9K empty carrier was purchased from Invitrogen. Sal I, EcoR I, and Not I were all purchased from Takara.

[0048] Example 1 1. Synthesis of COL17_pPIC9K expression plasmid The amino acids at positions 957-968, 1441-1482, 914-932, 914-932, and 914-932 of human type XVII collagen were sequentially combined to obtain collagen monomer fragment 1, as shown in SEQ ID NO.1. This was repeated twice to obtain a recombinant collagen single chain, as shown in SEQ ID NO.3, consisting of 216 amino acids. Codon optimization and chemical synthesis of the nucleotide sequence were commissioned to Nanjing Genscript Biotech Co., Ltd., and the optimized nucleotide sequence is shown in SEQ ID NO.4. Recombinant plasmid construction was also commissioned to Nanjing Genscript Biotech Co., Ltd.: the synthesized gene fragment was inserted between the EcoRI / Not I sites of the pPIC9K vector via EcoRI (Takara product number: 1611) and Not I (Takara product number: 1623) restriction enzyme sites to obtain the recombinant plasmid COL17_pPIC9K, as shown in the plasmid map. Figure 1 As shown.

[0049] 2. Construction of engineered Pichia pastoris strains The above recombinant plasmid (2-3 μg) was digested with Sal I (purchased from Takara) at 37℃ for 30 min, linearized, recovered, and electroporated into Pichia pastoris GS115 competent cells; the electroporated bacterial culture was plated on MD plates and incubated upside down at 28℃ for 2-5 days. Single colonies were selected to verify that the Pichia pastoris genetically engineered strain was obtained and named GS115-COL17.

[0050] 3. Modification of Pichia pastoris GS115 and Construction of New Engineered Strains like Figure 2 As shown, a knockout cassette targeting the Pichia pastoris GS115 YPS1 gene was constructed using homologous recombination technology. Precise knockout of the YPS1 gene was achieved through double crossover homologous recombination, yielding GS115-YPS1. Linearized plasmids were prepared using the same method and electroporated into GS115-YPS1 competent cells. The electroporated bacterial culture was plated on MD plates and incubated upside down at 28°C for 2–5 days. Single colonies were selected to verify the emergence of a new Pichia pastoris genetically engineered strain, named GS115-YPS1-COL17.

[0051] 4. Induced expression and identification of recombinant collagen Pick a single colony from an MD plate and inoculate it into 25 mL of BMGY medium. Incubate overnight at 28°C and 220 rpm. Take the colony during the logarithmic growth phase (approximately 16 hours, OD200). 600 =4-6) Centrifuge the bacterial culture at 4000 rpm at room temperature to collect the cells; after removing the supernatant, resuspend the cells in BMMY medium to OD=1 and induce expression (total culture medium volume 100 mL). Add anhydrous methanol to the culture medium every 24 h to a final concentration of 1.0%, and collect the expression supernatant after 72 h of induction. Perform SDS-PAGE detection and select the high expression level as the subsequent engineered bacteria.

[0052] 5. Fermentation expression and purification of COL17 protein ①Fermentation expression of COL17 Glycerol batch culture: Accumulating biomass to OD 600 Upon reaching the critical value of 30, glycerol-fed culture was initiated, with 50% glycerol (containing 12 mL / L PTM1) added at a rate of 4-6 mL / L / h, continuing the reaction until the OD value was reached. 600 =100±20; During the methanol induction phase, the methanol feed rate was gradually increased (12 mL of PTM1 per liter of methanol). For the first 0-12 hours, a low feed rate (2 mL / L / h) was used to avoid methanol toxicity. From 12-24 hours, the feed rate was increased to 4 mL / L / h, and DO fluctuations were monitored. From 24-96 hours, a high feed rate (6 mL / L / h) was used to maintain DO at 10%-20%, and induction continued for 96 hours to maximize protein secretion. The expression supernatant was collected and analyzed using SDS-PAGE. The results are as follows: Figure 3 The modified Pichia pastoris can effectively reduce protein degradation when expressing COL17.

[0053] ② Collection and purification of expression products The fermentation broth was centrifuged at 3000 g at room temperature, and the supernatant was collected. The supernatant was then diluted with purified water until the final solution conductivity was <5 mS / cm.

[0054] Cation exchange resin (Borglon, Diamond SP Mustang, catalog number AI0192) was used for separation and purification by chromatography. (I) Equilibration: 10mM PBS, pH 7.0 ± 0.1; wash with 3 column volumes and drain waste liquid; (II) Sample loading: After centrifugation, collect the supernatant of the fermentation broth and add it to the chromatography column, where it flows through the column resin by gravity; (III) Washing: 10 mM PBS, pH 7.0 ± 0.1; wash with 5 column volumes; (IV) Elution: 10 mM PBS, 0.5 M NaCl, pH 7.0 ± 0.1; use 3 column volumes for elution; (V) CIP cleaning: 500mM NaOH; flush with 5 column volumes.

[0055] Purified SDS-PAGE detection results are as follows Figure 4 As shown, the eluent was concentrated by ultrafiltration to obtain recombinant type XVII humanized collagen COL17 stock solution; it was filtered through a 0.22µm PES membrane into a sterile vial and then freeze-dried to obtain recombinant type XVII humanized collagen freeze-dried sponge.

[0056] Example 2 To screen for the optimal number of repetitions, under the same conditions as in Example 1, recombinant collagen single chains with 1, 2, 3, 4, 6, 8, and 10 repetitions were constructed and named COL17-1, COL17-2, COL17-3, COL17-4, COL17-6, COL17-8, and COL17-10, respectively (where COL17-2 is COL17 in Example 1). They were prepared using the same expression and purification conditions (same as in Examples 2-3) and their performance was tested according to the methods in Test Examples 1-3. The results are shown in Table 1.

[0057] Table 1 Performance comparison at different number of repetitions

[0058] Table 1 shows that the best overall performance was achieved with two repetitions, with the main band accounting for 93%. It remained stable after heating at 60℃ for 4 hours, forming a typical triple helix structure, and both cell adhesion and proliferation rates reached their peak (100%). The performance with three repetitions was similar to that with two repetitions (90% main band percentage, 98% adhesion rate, and 99% proliferation rate), also exhibiting good stability and biological activity. Therefore, the preferred number of repetitions in this invention is 2-3, with two repetitions being the most preferred. With fewer repetitions (one repetition), although the expression stability was highest (96% main band percentage), the biological activity was significantly lower. Because the monomer fragment contains only one set of functional domains and has a very small molecular weight (only 108 amino acids), it cannot form a stable triple helix structure, and the cell adhesion rate (72%) and proliferation rate (75%) are much lower than with two repetitions. This indicates that the "stability" of the sequence alone is insufficient to endow collagen with the necessary biological functions; therefore, one repetition cannot meet the needs of practical applications. When the number of repetitions is excessive (≥4 times), the proportion of the main band continuously decreases with the increase in the number of repetitions (from 85% in 4 repetitions to 55% in 10 repetitions), and the stability after heating at 60℃ for 4 hours also gradually deteriorates from "basically stable" to "significantly degraded." This is because excessively high repetition counts amplify the protease recognition sites and unstable local secondary structures hidden in the sequence, leading to non-specific degradation of the target protein during fermentation. Simultaneously, biological activity also decreases with increasing repetition count.

[0059] Example 3 To screen for the optimal fragment combination, under the same conditions as in Example 1 (all repeated twice), recombinant collagen single chains with the following different fragment combinations were constructed: (1) COL17-5F (this invention): 957-968 + 1441-1482 + 914-932 + 914-932 + 914-932 (i.e., the SEQ ID NO.1 fragment in Example 1); (2) COL17-Nat (natural continuous sequence): The natural continuous amino acid sequence of human type XVII collagen from position 914 to 968 is selected as SEQ ID NO.5: GHPGPPGPRGHKGEKGDKGDQGPKGDQGPPPGPRGHQGEQGPKGDQGPPGPRGHQGEQGPKGDQGPPGPRGHQGEQ; Repeat twice; (3) COL17-Rev (reverse splicing): 914-932 bits + 914-932 bits + 914-932 bits + 1441-1482 bits + 957-968 bits (reverse the splicing order) repeat 2 times; (4) COL17-A: Amino acids only at positions 914-932: SEQ ID NO.6: GHPGPPGPRGHKGEKGDKGD; Repeat twice; (5) COL17-B: Amino acids only at positions 957-968: SEQ ID NO.7: GPQGPKGDKGDPGQKGEMGTP; Repeat twice; (6) COL17-C: Amino acids only at positions 1441-1482; SEQ ID NO.8: GPRGHQGEQGPKGDQGPPGPRGHQGEQGPKGDQGPPGPR.

[0060] Repeat twice.

[0061] The same expression and purification conditions were used for preparation (same as in Example 1), and the performance was tested according to the methods of Test Examples 1-3. The results are shown in Table 2.

[0062] Table 2 Performance Comparison of Different Fragment Combinations

[0063] Table 2 shows that while COL17-A, COL17-B, and COL17-C, containing only single functional fragments, exhibit extremely high expression stability (93%–95% of the main band) due to their short sequences, their cell adhesion rate (60%–65%) and proliferation rate (63%–68%) are significantly lower than those of COL17-5F (100%) in this invention due to the lack of a complete functional domain combination of type XVII collagen. This indicates that retaining only a single functional fragment cannot form a complete biological functional conformation, and a single fragment cannot replace the synergistic effect of multiple fragments. Although COL17-Nat (natural continuous sequence repeated twice) retains the complete natural sequence, the main band accounts for only 52%, and significant degradation occurs after heating at 60°C. This is because the natural continuous sequence contains multiple protease recognition sites, which are further amplified after repetition, leading to a large amount of non-specific degradation of the target protein during fermentation, making industrial production difficult. The main band of COL17-Rev (reverse splicing) accounted for 82%, and slight degradation occurred after heating at 60°C. Its stability and activity were lower than those of COL17-5F of this invention, indicating that the order of fragment splicing has an important influence on the protein folding efficiency and exposure to protease attack sites.

[0064] Example 4 Collagen hemostatic dressings were prepared using the recombinant collagen obtained in Example 1. Recombinant XVII type humanized collagen lyophilized sponge 1.0%~2.0% (w / v), chitosan 0.5%~1.0% (w / v), glycerol or sorbitol 0.5%~2.0% (v / v), cross-linking agent (genipin / EDC) 0.01%~0.1% (w / v), water for injection to make up to 100%.

[0065] Preparation method: 1. Dissolve the recombinant XVII type humanized collagen freeze-dried sponge prepared in Example 1 in water for injection to prepare a 2.5% (w / v) collagen solution. Stir magnetically or at low speed (4°C) to ensure complete dissolution and avoid generating bubbles.

[0066] 2. Weigh out the chitosan, dissolve it in 1% acetic acid solution to prepare a 3.0% chitosan solution, and filter to remove insoluble matter.

[0067] 3. Mix the collagen solution and chitosan solution at a volume ratio of 8:2 and stir slowly until homogeneous; add glycerol or sorbitol to a final concentration of 0.5% and stir until homogeneous; add genipin (final concentration 0.01%) and stir for 30 minutes to allow it to react fully, and the product is ready.

[0068] Example 5 Collagen compound gel dressing was prepared using the recombinant collagen obtained in Example 1: It is divided into two parts, A and B. Part A is a sterile freeze-dried sponge containing 4 mg of recombinant type XVII humanized collagen. Part B consists of the following components by mass percentage: sodium hyaluronate 0.25%, carbomer 1%, propylene glycol 2.5%, glycerin 2%, methylparaben 0.05%, propylparaben 0.05%, and the remainder is purified water. Parts A and B should be mixed before use.

[0069] The preparation method is as follows: (1) Accurately weigh carbomer, sodium hyaluronate, glycerin, propylene glycol, methylparaben and propylparaben, stir for 1 hour, add purified water and dissolve completely.

[0070] (2) Accurately weigh the recombinant XVII type humanized collagen freeze-dried sponge.

[0071] (3) Filling under aseptic conditions, sealing and packaging, and irradiation sterilization result in V-type recombinant collagen compound gel dressing product.

[0072] Instructions for use: Bottles A and B can be aseptically and quickly mixed before use.

[0073] Example 6 Collagen liquid dressing was prepared using the recombinant collagen obtained in Example 1: The recombinant XVII type humanized collagen lyophilized sponge contains 5 mg / mL of collagen. The remaining components, by mass percentage, include: 0.05% sodium hyaluronate, 10% glycerol, 2% carbomer, and the remainder is purified water.

[0074] The preparation method is as follows: (1) Weigh carbomer, sodium hyaluronate and glycerin accurately, stir for 1 hour to dissolve completely.

[0075] (2) Accurately weigh the recombinant XVII type humanized collagen freeze-dried sponge, add purified water, and dissolve it completely.

[0076] (3) After mixing, fill the package under aseptic conditions, seal it, and sterilize it by irradiation to obtain the V-type recombinant collagen liquid dressing product.

[0077] How to use: After cleansing, apply evenly to the face and gently massage or pat until fully absorbed.

[0078] Comparative Example 1 Collagen hemostatic dressings were prepared using collagen obtained from COL17-Nat (same as Example 4).

[0079] Comparative Example 2 Collagen compound gel dressings were prepared using collagen obtained from COL17-Nat (same as Example 5).

[0080] Comparative Example 3 Collagen hemostatic dressings were prepared using collagen obtained from COL17-Rev (same as Example 4).

[0081] Comparative Example 4 Collagen compound gel dressings were prepared using collagen obtained from COL17-Rev (same as Example 5).

[0082] Test Example 1 The properties of the recombinant collagen freeze-dried collagen sponge obtained in Example 1 were tested: 1. Thermal stability Recombinant type XVII humanized collagen lyophilized sponge was prepared into a 10 mg / mL solution with ultrapure water, filtered through a 0.22 μm filter membrane, and then aliquoted into sterile centrifuge tubes. The tubes were heated at 60 °C for 1 h, 2 h, 3 h, and 4 h, and samples were taken for SDS-PAGE analysis.

[0083] The stability results of the recombinant XVII type humanized collagen freeze-dried sponge are as follows: Figure 5As shown, the results indicate that the treated recombinant XVII type humanized collagen freeze-dried sponge still maintains structural and purity stability, with no significant difference from 0h; the results also show that the protein structure was not destroyed at 60℃, and no serious degradation or diffusion occurred, proving that the recombinant XVII type humanized collagen freeze-dried sponge has extremely high thermal stability.

[0084] 2. Acid and alkali tolerance stability test The stability of recombinant XVII type humanized collagen lyophilized sponge was tested under different pH conditions, and the results are as follows: Figure 6 As shown, the results indicate that the recombinant XVII type humanized collagen freeze-dried sponge has good pH suitability, maintains strong stability at pH 6-8, and has good clinical pH suitability.

[0085] 3. The specific implementation method of the cell proliferation experiment is as follows: Recombinant humanized collagen COL17 solution and BSA solution of the same concentration were prepared as experimental groups. PBS solution was added to the control group. Then, 100 μL of solution was added to each well of a 96-well plate. Each sample was coated to prepare 3 wells. The plates were incubated in a 5% CO2 incubator at 37°C for 2 h. The excess coating solution was removed and the 96-well plates were refrigerated at 2-8°C overnight.

[0086] L929 cells were digested and counted. The suspension was diluted with growth medium and seeded into 96-well plates at a density of 2000 cells / well (100 μL per well). The seeding time was recorded as 0 h. The cells were incubated at 37°C for 11 h in a 5% CO2 incubator. After incubation, 10 µL of CCK-8 solution was added to each well, and the cells were incubated again at 37°C for 1 h. The absorbance was then measured at 450 nm.

[0087] 4. The specific implementation method of the cell adhesion experiment is as follows: Coating: Recombinant type XVII humanized collagen COL17 solution and BSA solution of the same concentration were prepared as the experimental group. PBS solution was added to the control group. 100 μL of solution was added to each well of a 96-well plate. Three wells were prepared for each sample coating. The plates were incubated at 37°C for 2 h in a 5% CO2 incubator.

[0088] Washing: Remove excess coating solution from the wells and wash twice with PBS solution.

[0089] Cell counting: Cells were resuspended in culture medium and counted. The cell count was diluted to a final concentration of 2 × 10⁻⁶. 5 per mL.

[0090] Assay: The cell suspension was aspirated and added to each well of a 96-well plate, 100 μL per well. After incubation at 37°C for 4 h in a 5% CO2 incubator, the culture medium was discarded. The cells were gently washed three times with PBS to remove any unadhered cells. Fresh culture medium was then added and cultured for 2 h. After incubation, 10 µL of CCK-8 solution was added to each well, and the plate was incubated for another 1 h. The absorbance was then measured at 450 nm.

[0091] Observation of cell adhesion quantity and morphology: (1) Under a 100x light microscope, the quantity and morphology of L929 cells when they were first seeded into the well plates of the three samples were observed and compared. (2) After incubation for 4 h, under the same magnification conditions, the changes in cell morphology and the number of adherent cells at different times were observed and compared.

[0092] 5. The specific implementation method of the cell migration experiment is as follows: Coating: Prepare recombinant type XVII humanized collagen solution and BSA solution of the same concentration as the experimental group. Add PBS solution to the control group. Add 1.5 mL of solution to each well of a marked 6-well plate. Coat two wells for each sample. Incubate at 37°C for 2 h in a 5% CO2 incubator.

[0093] Washing: Remove excess coating solution from the wells and wash twice with PBS solution.

[0094] Cell counting: Cells were resuspended in culture medium and counted. The cell count was diluted to a final concentration of 3 × 10⁻⁶. 5 per mL.

[0095] Detection: Add 1 mL of the cell suspension to each well of a 6-well plate and incubate at 37°C for 20-24 hours in a 5% CO2 incubator until 95%-100% confluence. Using a 1 mL pipette tip, gently push downwards along the horizontal marking line to create a scratch (approximately 1.0-1.5 mm wide). Wash twice gently with PBS to remove the scratched cells, then add 2 mL of fresh culture medium and incubate at 37°C in a 5% CO2 incubator. Observe the healing of the cell scratches under a microscope at 0 h and 16 h, using the intersection of the horizontal and vertical scratches as the focal point, and photograph the cells under a 40x microscope for analysis.

[0096] Experimental results are as follows Figure 7 The results showed that recombinant type XVII humanized collagen COL1-3 promoted cell proliferation, cell adhesion, and cell migration.

[0097] 6. Safety The testing should be conducted according to GB / T16886.5-2017, and the steps are as follows: The potential cytotoxic effects of recombinant humanized type XVII collagen were tested using in vitro cultured mammalian L-929 cells. Recombinant collagen (1 mg / mL, 2 mg / mL, 4 mg / mL) and control samples were placed in MEM medium containing 10% fetal bovine serum and incubated at 37°C for 24 hours. After extraction, 96-well plates (103 wells) cultured for 24 hours were... 4 Remove the culture medium from each cell (cells / well), replace it with the appropriate extraction solution, and incubate in a cell culture incubator (37℃, 5% CO2, >90% humidity) for 24 hours.

[0098] After culture, cell morphology and cell lysis were observed under a microscope, and the cytotoxicity value of the test sample was determined using the CCK8 assay. Results are as follows: Figure 8 As shown, the results indicate that the blank control group and cells maintained normal morphology throughout the experiment and showed no cytotoxic reaction. After incubating the cells at a concentration of 4 mg / mL for 24 hours, the cell morphology remained basically intact and the cell viability was close to 100%. This shows that recombinant type XVII humanized collagen had no cytotoxic effect under the CCK8 cytotoxicity assay conditions.

[0099] Test Example 2 An evaluation experiment on the healing of diabetic wounds was conducted using the products of Example 5 and Comparative Examples 2 and 4: Eighty healthy SPF-grade SD rats, weighing between 230-250g, were used. They were acclimatized for one week before the experiment, housed at a temperature of 20±3℃ and a relative humidity of 55±10%, with free access to food and water. Before modeling, each rat was fasted for 12 hours and injected with 1% STZ solution (prepared with citrate buffer) at a dose of 50mg / kg. Thirty minutes after injection, food and water intake were no longer restricted. Blood glucose was measured from the tail vein 72 hours after injection. A blood glucose level ≥16.7mM indicated successful modeling; rats that did not successfully model were excluded from the experiment. Rats with successful modeling were divided into four groups of 15 each.

[0100] A 2cm diameter circle was cut on the back of a rat to obtain a skin lesion. The collagen compound gel dressings obtained in Example 5, Comparative Example 2, and Comparative Example 4 were applied to the skin lesion twice daily for four weeks. The control group was treated with a phosphate buffer solution at pH 7.0. The diameter of the skin lesion was measured to confirm the wound healing effect. The results are as follows Figure 9 As shown, by Figure 9It can be seen that the collagen compound gel dressing prepared in Example 5 of the present invention has a good effect on promoting wound healing, and the wound healing rate reaches 99% after 28 days. Compared with the control group, the gels of Comparative Examples 2 and 4 are both effective compared with the blank control, and the effect of Comparative Example 2 is better than that of Comparative Example 4; but the effect is worse than that of Example 5.

[0101] Test Example 3 Hemostatic effect of the products from Example 4 and Comparative Examples 1 and 3: Fifty 5-week-old Kunming mice, weighing 25±2g, were randomly divided into 5 groups, half male and half female, with 10 mice in each group. Each mouse was anesthetized by injecting 3% sodium pentobarbital at a dose of 30mg / kg. The mice were then fixed, and the tails were cut off 1cm from the end with surgical scissors. Any bleeding was treated immediately with hemostatic materials. The blank group used physiological saline. Group 4, Comparative Example 1, and Comparative Example 3 used the hemostatic dressings of Example 4, Comparative Example 1, and Comparative Example 3, respectively. The positive control group was treated with Yunnan Baiyao powder. Timing was started from the tail cut, and bleeding was considered stopped after 20 minutes. The results are shown in Table 3.

[0102] Table 3. Effects of different products on hemostasis time in mice

[0103] As shown in Table 3, the hemostasis time of Example 4 was the shortest, averaging 135 seconds, significantly better than the positive control group and all comparative groups. Compared with commercially available Yunnan Baiyao, this indicates that the recombinant collagen of the present invention has excellent procoagulant activity. The hemostasis time of Comparative Example 1 was 162 seconds, faster than the blank control and positive control, but slower than Example 4 of the present invention. This result indicates that although COL17-Nat retains the core functional domain of type XVII collagen and possesses certain biological activity, its extremely poor expression stability and the presence of a large number of degradation fragments in the fermentation product result in a low proportion of effective components in the actual product, severely affecting its procoagulant effect. The hemostasis time of Comparative Example 3 was 171 seconds, the worst among the three test samples, only slightly better than the positive control group. This is consistent with its activity data. Due to the change in the fragment splicing order, the spatial conformation of some functional domains of COL17-Rev is affected, resulting in reduced biological activity, and therefore it cannot effectively promote platelet aggregation and coagulation cascade reactions.

[0104] Test Example 4 In this embodiment, the COL17 lyophilized sponge prepared in Example 1 was subjected to circular dichroism (CD) chromatography. The structural characterization of collagen in this invention employs circular dichroism spectroscopy, a method commonly used in the field. The CD characteristic of the triple helix structure of collagen generally exhibits a positive absorption peak near 223 nm and a negative absorption peak near 195 nm; the positions of these absorption peaks shift with changes in amino acid sequence and length. A 0.5 mg / mL solution of the COL17 lyophilized sponge prepared in Example 3 was prepared for CD detection. The results are as follows... Figure 10 As shown, the maximum characteristic positive peak is at approximately 223 nm, and a negative peak appears at less than 200 nm (198 nm), verifying that the recombinant collagen of the present invention has a triple helix structure.

[0105] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A recombinant type XVII humanized collagen, characterized in that, The recombinant type XVII humanized collagen includes a recombinant collagen single chain; wherein the recombinant collagen single chain is composed of collagen monomer fragments as shown in SEQ ID NO.1 repeated 2 to 10 times.

2. The recombinant type XVII humanized collagen according to claim 1, characterized in that, The recombinant collagen single chain is obtained by cutting and splicing the human type XVII collagen α1 chain; wherein the amino acid sequence of the human type XVII collagen is shown in SEQ ID NO.

2.

3. The recombinant type XVII humanized collagen according to claim 2, characterized in that, The collagen monomer fragment is obtained by sequentially combining amino acids at positions 957-968, 1441-1482, 914-932, 914-932, and 914-932 of human type XVII collagen, wherein the amino acid sequence of the human type XVII collagen is shown in SEQ ID NO.

2.

4. The recombinant type XVII humanized collagen according to claim 3, characterized in that, The amino acid sequence of the recombinant collagen single chain is shown in SEQ ID NO.

3.

5. The recombinant type XVII humanized collagen according to claim 3, characterized in that, The nucleotide sequence encoding the recombinant collagen single chain is shown in SEQ ID NO.

4.

6. A method for preparing recombinant collagen single chains, characterized in that, Includes the following steps: Step 1: Design collagen sequences The amino acids at positions 957-968, 1441-1482, 914-932, 914-932, and 914-932 of human type XVII collagen were sequentially combined to obtain collagen monomer fragments. The collagen monomer fragments shown in SEQ ID NO.1 were repeated 2 to 10 times to obtain recombinant collagen single chains. Step 2, constructing the carrier Based on the recombinant collagen single-chain, the corresponding nucleotide sequence was synthesized to construct a recombinant plasmid; Step 3: Transformation and Cultivation The recombinant plasmid was transferred into host cells, engineered strains were screened, and fermentation culture was performed to induce the expression of the target protein. Step 4: Harvesting and Purification The fermentation broth was collected, the expressed protein product was harvested, and purified to obtain the target collagen.

7. The preparation method according to claim 6, characterized in that, In step two, the plasmid is selected for prokaryotic expression, specifically the pET series vector, pGEX series vector, pBAD series vector, or pUC series vector; or the plasmid is selected for eukaryotic expression, specifically the pcDNA series vector, pCMV series vector, pIRES series vector, pEGFP series vector, pYES series vector, or pPIC series vector.

8. The preparation method according to claim 6, characterized in that, In step three, the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell.

9. The use of a stable type XVII recombinant collagen prepared by the method according to any one of claims 6-8 in the preparation of collagen-containing products.

10. The application according to claim 9, characterized in that, The collagen products mentioned are cosmetics, medical devices, or pharmaceuticals.

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