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

Abstract

The present application belongs to the technical field of protein engineering and genetic engineering, and particularly relates to a recombinant collagen XVII and a preparation method and application thereof. Specifically, a brand new recombinant collagen is designed through screening, replacement and splicing, the nucleotide sequence of the recombinant collagen is optimized according to the codon bias of Pichia pastoris, a transmembrane peptide is introduced into the amino terminal of the sequence, a corresponding genetic engineering yeast strain is constructed, and the recombinant collagen is successfully prepared through fermentation. The amino acid sequence of the recombinant collagen has a homology of 100% with the corresponding region of natural human collagen XVII, and there is no immunogenicity risk caused by sequence difference. Meanwhile, the endotoxin content of the protein can be controlled through a purification process, so that no endotoxin residue is ensured. The recombinant collagen prepared by the above technical scheme has more excellent transdermal performance and cell proliferation activity, and has better stability, and therefore has good practical application value.

Description

Technical Field

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

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Collagen, a key structural component of the extracellular matrix, is the most abundant and widely distributed functional structural protein in mammals, accounting for about one-third of total protein. It is widely found in tissues such as skin, bone, tendons, ligaments, and mucous membranes. Due to its excellent biological functions, low immunogenicity, and biodegradability, it has wide applications in medicine, tissue engineering, food, and cosmetics. Currently, 29 types of collagen have been identified. Type XVII collagen, a transmembrane collagen among non-fibroblast collagens, possesses high biocompatibility and good efficacy. It is a key molecule in preventing the aging of hair follicle stem cells and skin stem cells, and plays a significant role in maintaining the dryness characteristics of hair follicles, promoting hair regeneration, and maintaining skin homeostasis and a youthful state.

[0004] Recombinant collagen has broad application prospects due to its ability to be produced on a large scale and with high purity, and its ability to effectively avoid pathogen risks. However, the recombinant preparation and application of type XVII collagen in current technologies face many challenges. Type XVII collagen is 1497 amino acids long and includes intracellular, transmembrane, and extracellular domains. It is composed of 16 non-triple-helix regions and 15 triple-helix regions arranged alternately, making in vitro recombinant expression difficult. Current technologies use expression systems such as E. coli, selecting different truncated sequences or splicing combinations for expression. However, the E. coli expression system lacks post-translational modification activity, and regardless of the expression system used, the collagen prepared as a skincare ingredient has a natural micelle structure and high molecular weight, making it difficult for it to penetrate the skin barrier and reach the dermis to exert its effects.

[0005] In hair growth promotion applications, the effective delivery of type XVII collagen is crucial. While commonly used microneedling methods can improve the transdermal delivery efficiency of large molecular active ingredients, they are highly destructive to the skin, potentially causing local tissue damage, pain, cross-infection, and allergic reactions. Some studies have attempted to use ionic liquids to address the large molecule delivery problem, but their formulations generally exhibit strong irritant and sensitizing properties, resulting in poor skin compatibility. Furthermore, the preparation processes of some recombinant collagens are complex, time-consuming, and costly, requiring multiple complex gene manipulations and cell culture and separation steps, and demanding stringent equipment and environmental requirements, hindering large-scale production applications. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a recombinant type XVII collagen, its preparation method, and its applications. The recombinant type XVII collagen prepared by this invention exhibits excellent transdermal permeability and cell proliferation-promoting properties, and can be efficiently and stably expressed in yeast. Based on the above research results, this invention is thus completed.

[0007] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0008] A first aspect of the present invention provides a recombinant type XVII collagen, said recombinant type XVII collagen comprising:

[0009] (a1) The amino acid sequence shown in SEQ ID NO.1;

[0010] (a2) An amino acid sequence derived from SEQ ID NO.1 that has been substituted, deleted or added one or more amino acids and has type XVII collagen activity;

[0011] (a3) Proteins encoded by other genes that have more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and have the protein activity shown in SEQ ID NO.1.

[0012] Furthermore, the recombinant type XVII collagen is also modified with membrane-penetrating peptides.

[0013] In a second aspect, the present invention provides a polynucleotide capable of encoding the aforementioned recombinant type XVII collagen.

[0014] A third aspect of the present invention provides a recombinant expression vector comprising the polynucleotide.

[0015] A fourth aspect of the present invention provides a host cell containing the recombinant expression vector or having the polynucleotide integrated into a chromosome or being able to express the recombinant type XVII collagen.

[0016] A fifth aspect of the present invention provides a method for preparing the recombinant type XVII collagen, comprising: culturing the host cells to express the recombinant type XVII collagen; and isolating and purifying the recombinant type XVII collagen.

[0017] A sixth aspect of the present invention provides the use of the above-described recombinant type XVII collagen in the preparation of health foods, cosmetics, medical aesthetic products, or pharmaceuticals.

[0018] A seventh aspect of the present invention provides a scalp care product comprising the above-described recombinant type XVII collagen.

[0019] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0020] The above-mentioned technical solution designs a novel recombinant collagen protein through screening, replacement, and splicing. Its nucleotide sequence is optimized using Pichia pastoris codon preference, and a transmembrane peptide is introduced at the amino terminus of the sequence to construct a corresponding genetically engineered yeast strain. The recombinant collagen protein is then successfully prepared through fermentation. Its amino acid sequence shows 100% homology with the corresponding region of natural human type XVII collagen, eliminating the risk of immunogenicity due to sequence differences. Furthermore, the endotoxin content of this protein can be controlled through purification processes to ensure no endotoxin residue. Experimental results show that the recombinant collagen protein prepared using the above-mentioned technical solution exhibits superior transdermal properties and cell proliferation activity compared to commercially available recombinant collagen proteins, while also demonstrating better stability. Therefore, the above-mentioned technical solution effectively solves the technical problem of difficult transdermal collagen production and can further enhance its bioactivity and efficacy, thus possessing significant practical application value. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 SDS-PAGE results for protein expression in COL1510 shake flask fermentation (samples taken from 0h to 96h).

[0023] Figure 2 SDS-PAGE results of protein expression in TAT-COL1510 shake flask fermentation (samples taken from 0h to 96h).

[0024] Figure 3 SDS-PAGE results of protein expression in TAT-COL1510 and COL1510 5L fermenters (sampled and detected from 0h to 96h).

[0025] Figure 4 The results of TAT-COL1510 SDS-PAGE salting out at a gradient concentration of 20%-70% ammonium sulfate are shown.

[0026] Figure 5 The results of COL1510 SDS-PAGE salting out at a gradient concentration of 20%-70% ammonium sulfate are shown. Detailed Implementation

[0027] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. 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 invention pertains.

[0028] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0029] As mentioned earlier, most collagen products currently on the market share the common problem of large molecular weight. This characteristic makes it difficult for them to break through the skin's epidermal barrier and effectively penetrate into the superficial dermis, thus limiting their full biological function.

[0030] In view of this, in a typical embodiment of the present invention, a recombinant type XVII collagen is provided, the recombinant type XVII collagen comprising:

[0031] (a1) The amino acid sequence shown in SEQ ID NO.1;

[0032] (a2) An amino acid sequence derived from SEQ ID NO.1 that has been substituted, deleted or added one or more amino acids and has type XVII collagen activity;

[0033] (a3) Proteins encoded by other genes that have more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO.1 and have the protein activity shown in SEQ ID NO.1.

[0034] The proteins shown in (a1)-(a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0035] Furthermore, the recombinant type XVII collagen significantly enhances transdermal performance by modifying a membrane-penetrating peptide, which can be modified at the carboxyl terminus (C-terminus) or amino terminus (N-terminus) of the recombinant type XVII collagen, with the amino terminus being the preferred modification site.

[0036] Selection of membrane-penetrating peptides: The membrane-penetrating peptides that can be selected include YARA (amino acid sequence as shown in SEQ ID NO.3), SPACE (amino acid sequence as shown in SEQ ID NO.4), TAT (amino acid sequence as shown in SEQ ID NO.5) and ANTP (amino acid sequence as shown in SEQ ID NO.6); TAT is further preferred.

[0037] Therefore, in one specific embodiment of the present invention, the amino acid sequence of the recombinant type XVII collagen is as shown in SEQ ID NO.7.

[0038] In another specific embodiment of the present invention, a polynucleotide is provided, which is capable of encoding the above-mentioned recombinant type XVII collagen. Specifically, the nucleotide sequence of the polynucleotide is shown in SEQ ID NO.2 or SEQ ID NO.8.

[0039] The polynucleotide can be DNA, such as cDNA, genomic DNA, or recombinant DNA, etc., and is not specifically limited here.

[0040] In another specific embodiment of the present invention, a recombinant expression vector is provided, the recombinant expression vector comprising the polynucleotide.

[0041] According to the present invention, the recombinant expression vector is obtained by effectively linking the aforementioned polynucleotides to an expression vector. The expression vector is any one or more of a viral vector, plasmid, bacteriophage, kinase, or artificial chromosome. The viral vector may include adenovirus vector, retrovirus vector, or adeno-associated virus vector. The artificial chromosome includes bacterial artificial chromosomes, bacteriophage P1-derived vectors, yeast artificial chromosomes, or mammalian artificial chromosomes. In another specific embodiment of the present invention, the expression vector is a plasmid, specifically any plasmid vector from the pPIC series, and more specifically, the expression vector is the pPIC9K plasmid.

[0042] In another specific embodiment of the present invention, a host cell is provided, wherein the host cell contains the recombinant expression vector or the chromosome is integrated with the polynucleotide or is capable of expressing the recombinant type XVII collagen.

[0043] Furthermore, the host cell is a bacterial cell, a fungal cell, a mammalian cell, or an insect cell.

[0044] Furthermore, the fungal cells are yeast cells, and the yeast may be Pichia pastoris (…). Pichia pastorisExamples of strains that can express the same information include *Pichia pastoris* X33, GS115, SMD1168, KM71, or KM71H. However, this invention does not limit this to a single strain, and other *Pichia pastoris* strains with equivalent expression functions are also within the scope of protection of this invention.

[0045] In another specific embodiment of the present invention, a method for preparing the recombinant type XVII collagen is provided, comprising: culturing the host cells to express the recombinant type XVII collagen; and isolating and purifying the recombinant type XVII collagen.

[0046] As a specific implementation of the separation and purification steps of this invention, it may include, but is not limited to, the following steps: plate and frame filtration (for removing solids such as cell residues in the fermentation system), hollow fiber membrane clarification (for further removing macromolecular impurities, colloidal substances, etc. in the system), ultrafiltration membrane concentration and replacement (to achieve concentration of recombinant collagen and replacement of the buffer system), salting out (to purify recombinant collagen and remove impurities), ultrafiltration membrane desalting (to remove salt from the system and optimize product purity), and freeze-drying (to obtain a stable solid product of recombinant collagen).

[0047] It should be noted that the specific parameters of the above separation and purification steps (such as filtration pore size, salting-out conditions, lyophilization curve, etc.) can be adaptively adjusted according to the molecular weight, isoelectric point, and other characteristics of the recombinant collagen. No specific limitations are made here.

[0048] In another specific embodiment of the present invention, the above-mentioned recombinant type XVII collagen is provided for use in the preparation of health foods, cosmetics, medical aesthetic products or pharmaceuticals.

[0049] The aforementioned health food is a type of food that shares common characteristics with general foods. It can regulate the body's functions and is suitable for consumption by specific groups of people, but it is not intended to treat diseases.

[0050] In this invention, the cosmetic product can be applied to the skin to achieve the effect of promoting cell proliferation. Therefore, the cosmetic product in this invention can be a skin care product.

[0051] Meanwhile, in this invention, the cosmetic dosage form can be an aqueous solution, emulsion, ointment, etc. Furthermore, other cosmetic categories derived and prepared based on the above-mentioned basic cosmetic categories, such as facial cleanser, sunscreen, toner, face cream, eye cream, essence water (lotion), hand cream (ointment), body lotion, and face mask, are also within the scope of protection of this application.

[0052] In this invention, the medical aesthetic products refer to products used in the field of medical aesthetics, and are not the same as ordinary daily skincare products or beauty salon skincare products. Some commonly used medical aesthetic products include medical aesthetic masks and dressings, etc. No specific limitations are made here.

[0053] In particular, the recombinant type XVII collagen in this invention can promote the proliferation of hair follicle-related cells (such as human hair papilla cells), thereby promoting hair growth. Therefore, it can be used as a scalp care product (such as a scalp care hair growth product), etc., without being specifically limited here.

[0054] In this invention, the medicine can be a medical device product. Specifically, the medical device product can be a topical gel, spray, or dressing containing the above-mentioned recombinant type XVII collagen.

[0055] In another specific embodiment of the present invention, a scalp care product is provided, the scalp care product comprising the above-mentioned recombinant type XVII collagen.

[0056] The present invention will be further described below with reference to embodiments. The present invention will be further illustrated by way of embodiments, but this does not limit the present invention to the scope of the embodiments described. Unless otherwise specified, the experimental methods used in the embodiments and comparative examples are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0057] The culture medium used for host bacterial culture and recombinant collagen expression in this embodiment of the invention has the following components:

[0058] 1. YPD medium

[0059] The amount of each component added in each liter (1L) of YPD medium is: 10 g yeast extract, 20 g peptone, and 20 g glucose. The preparation method of the medium is as follows: dissolve the above components in deionized water, make up to 1L, and sterilize before use.

[0060] 2. BMGY medium

[0061] The amounts of each component added per liter (1L) of BMGY medium are as follows: yeast extract 10g, peptone 20g, dipotassium hydrogen phosphate (K2HPO4) 3g, potassium dihydrogen phosphate (KH2PO4) 11.8g, yeast nitrogen base (YNB) 3.4g, ammonium sulfate ((NH4)2SO4) 10g, biotin 4×10 -4 10 grams of glycerin; dissolve the above components in deionized water, stir thoroughly until completely dissolved, and then bring the volume to 1L. After sterilization, it is ready for use.

[0062] 3. BMMY medium

[0063] The following components were added per liter (1L) of BMMY medium: yeast extract 10g, peptone 20g, dipotassium hydrogen phosphate (K2HPO4) 3g, potassium dihydrogen phosphate (KH2PO4) 11.8g, yeast nitrogen basal (YNB) 3.4g, ammonium sulfate ((NH4)2SO4) 10g, and biotin 4×10⁻⁶. -4 10 ml of methanol; except for methanol, dissolve the remaining components in deionized water and bring the volume to 1 L. After sterilization, wait for the culture medium temperature to drop to room temperature, then aseptically add methanol and mix well to avoid methanol volatilization or reaction at high temperature.

[0064] Example 1: Sequence design of recombinant collagen

[0065] This embodiment aims to provide a biologically active recombinant collagen sequence, and the specific design process is as follows:

[0066] Sequence source analysis: Based on the amino acid sequence of natural human type XVII collagen α1 chain protein (GenBank sequence number NP_000485.3), bioinformatics analysis was used to resolve the amino acid sequence characteristics (such as repetitive sequence units and domain distribution) of this natural collagen and the functional active sites (such as cell binding sites and enzymatic sites) reported in published literature.

[0067] Sequence optimization design: Based on the above analysis results, the basic sequence was screened (retaining the core active site), amino acid residues were replaced (optimizing protein stability and solubility), and sequence fragments were spliced ​​(removing redundant fragments) to finally obtain a target amino acid sequence, which corresponds to SEQ ID NO.1 in the sequence listing of this invention, and the recombinant collagen was named COL1510.

[0068] Example 2: Construction of an expression system containing recombinant collagen gene

[0069] This embodiment aims to construct a Pichia pastoris expression system that can efficiently express recombinant collagen COL1510. The specific steps are as follows:

[0070] Nucleotide sequence optimization: based on Pichia pastoris ( Pichia pastoris The codon usage preference of the nucleotide sequence encoding the recombinant collagen COL1510 described in Example 1 was optimized (to improve codon fit and enhance protein expression). The optimized nucleotide sequence corresponds to SEQ ID NO.2 in the sequence listing of this invention.

[0071] Gene synthesis and vector cloning: GenScript Biotech Ltd. was commissioned to synthesize the whole gene from the optimized nucleotide sequence to obtain the target gene fragment. The target gene fragment and the Pichia pastoris expression vector pPIC9K were double-digested with restriction endonucleases EcoRI and NotI. After recovering the digestion products by agarose gel electrophoresis, the target gene fragment was cloned into the EcoRI-NotI restriction sites of the pPIC9K vector using DNA ligase to construct the recombinant expression vector, named pPIC9K-COL1510.

[0072] Vector Validation and Host Transformation: The constructed recombinant expression vector pPIC9K-COL1510 was validated by DNA sequencing. The sequencing results showed that the target gene sequence was correct and the insertion direction was correct. The validated recombinant expression vector pPIC9K-COL1510 was linearized using the restriction endonuclease SalI, and the linearized vector was introduced into Pichia pastoris GS115 via electroporation. P. pastoris Recombinant transformants were obtained in GS115 host cells.

[0073] Screening for high-copy-count strains: The recombinant transformants were inoculated onto selection medium containing genimycin G418. By gradually increasing the G418 concentration (e.g., 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL), high-copy-count recombinant Pichia pastoris strains with stronger drug resistance were obtained and named […]. P. pastoris GS115 / pPIC9K-COL1510.

[0074] Example 3: Shake-flask fermentation of recombinant collagen engineered yeast strain

[0075] This embodiment aims to verify the expression ability of the high-copy strain constructed in Example 2 for recombinant collagen COL1510 through shake-flask fermentation experiments. The specific fermentation steps are as follows:

[0076] Seed culture: High-copy strains obtained in Example 2 were picked from solid culture medium. P. pastoris A single colony of GS115 / pPIC9K-COL1510 was inoculated into an Erlenmeyer flask containing 20 mL of YPD medium and placed in a shaker. The flask was then shaken and cultured for 18 h at 30°C and 220 rpm to obtain the seed culture.

[0077] Cell amplification culture: The above seed culture was transferred to an Erlenmeyer flask containing 50 mL of BMGY medium at an inoculation rate of 10% (v / v), and cultured with shaking at 30℃ and 220 rpm for 24 h to achieve cell amplification.

[0078] Induced expression culture: The amplified bacterial culture was placed in a centrifuge tube and centrifuged at 8000 rpm for 10 min to collect the bacterial cells. The bacterial cells were washed twice with sterile physiological saline to remove residual culture medium. The bacterial cells were then resuspended in 100 mL of BMMY induction medium. The suspension was placed in a shaker and induced at 25 °C and 220 rpm. Methanol was added to the culture medium every 24 h to a final concentration of 1% (v / v). The total induction time was 96 h.

[0079] SDS-PAGE validation of recombinant collagen COL1510 expression: SDS-PAGE protein electrophoresis analysis was performed on the fermentation supernatant. The electrophoresis results are as follows: Figure 1 As shown in the figure. The results show that a distinct single protein band appears at a position of approximately 35 kDa, which is the theoretical molecular weight, indicating that the target protein COL1510 was successfully expressed.

[0080] Example 4: Construction and Protein Expression Verification of a High-Copyright Strain Containing Membrane-Penetrating Peptides for Recombinant Collagen

[0081] This embodiment aims to optimize the transdermal performance of recombinant collagen by fusing a membrane-penetrating peptide, construct the corresponding high-copy-count expression strain, and verify the protein expression. The specific steps are as follows:

[0082] Design of recombinant collagen sequence containing transdermal peptide: The transdermal peptide TAT (amino acid sequence corresponding to SEQ ID NO.5 in the sequence listing of this invention) was selected. Based on the codon preference of Pichia pastoris, the coding sequence of the transdermal peptide was optimized, and the optimized transdermal peptide coding sequence was respectively connected to the 5' end (i.e. N end) of the COL1510 coding sequence described in Example 1 to obtain the coding sequence of the fusion recombinant collagen, named TAT-COL1510 (amino acid sequence corresponding to SEQ ID NO.7 in the sequence listing of this invention).

[0083] Construction of recombinant expression vector and screening of strains: Following the vector construction method in Example 2, the coding sequence of the above-mentioned fusion recombinant collagen was cloned into the pPIC9K vector via the EcoRI-NotI restriction site to construct the recombinant expression vector, named pPIC9K-TAT-COL1510; after verifying the sequence was correct by DNA sequencing, the recombinant vector was linearized with SalI and electroporated. P. pastoris GS115 host cells were used to select high-copy recombinant Pichia pastoris strains using genimycin G418, and these strains were named... P. pastoris GS115 / pPIC9K-TAT-COL1510.

[0084] SDS-PAGE verification of recombinant collagen expression containing membrane-penetrating peptides: The supernatant from shake-flask fermentation of the above-mentioned high-copy strain (fermentation method as described in Example 3) was collected, and the supernatant was analyzed by SDS-PAGE protein electrophoresis. The electrophoresis results are as follows: Figure 2 As shown in the figure. The results show that a distinct single protein band appears at a position of approximately 35 kDa, which is the theoretical molecular weight, indicating that the target fusion protein TAT-COL1510 was successfully expressed.

[0085] Example 5: Fermentation preparation of recombinant collagen COL1510 and recombinant collagen TAT-COL1510 containing membrane-penetrating peptides.

[0086] This embodiment aims to achieve efficient expression of recombinant collagen COL1510 and recombinant collagen TAT-COL1510 containing membrane-penetrating peptides by scaling up the culture of recombinant engineered bacteria in a 5L fermenter.

[0087] The culture medium and feed formulation used for fermentation in this embodiment are as follows:

[0088] BMG liquid culture medium: 10g yeast extract, 20g yeast peptone, 3g dipotassium hydrogen phosphate, 11.8g potassium dihydrogen phosphate, 4×10⁻⁶ biotin. -3 12.5 grams of glycerin and 12.5 grams of glycerol are dissolved in deionized water and stirred thoroughly until completely dissolved. The solution is then brought to a final volume of 1 L and sterilized before use.

[0089] FM22 fermentation medium: 2 g yeast extract, 2 g yeast peptone, 42.9 g potassium dihydrogen phosphate, 1.0 g calcium sulfate dihydrate, 5.0 g ammonium sulfate, 14.3 g potassium sulfate, 11.7 g magnesium sulfate heptahydrate, 40.0 g glycerol, 0.5 g defoamer, and 5.0 mg biotin. Dissolve the above components in deionized water, stir thoroughly until completely dissolved, and bring the volume to 1 L. After sterilization, set aside. After cooling to 25-30℃, add trace element stock solution PTM1 at a ratio of 4.35 mL / L.

[0090] Trace element stock solution PTM1: 6 g copper sulfate, 0.08 g sodium iodide, 3 g manganese sulfate, 14.9 g sodium molybdate, 0.02 g boric acid, 0.5 g cobalt chloride, 20 g zinc chloride, 65 g ferrous sulfate heptahydrate, 0.2 g biotin, and 5 mL sulfuric acid. Dissolve the above components in deionized water, stir thoroughly until completely dissolved, and then bring the volume to 1 L. Filter through a 0.22 μm filter membrane for sterilization, seal and store at 4°C for later use.

[0091] Glycerin replenishment: 50% (m / m) glycerin aqueous solution, autoclaved at 121℃ for 20 minutes, cooled to room temperature, added to PTM1 stock solution at a ratio of 12 mL / L, mixed well and stored at 4℃.

[0092] Methanol feed: Add 100% (v / v) methanol to PTM1 mother liquor at a ratio of 12 mL / L, mix well, seal and store at 4℃.

[0093] Seed liquid preparation

[0094] Primary seed culture: COL1510 and TAT-COL1510 recombinant collagen strains were inoculated into BMG liquid medium and cultured with shaking at 30℃ and 220 rpm until the OD reached 5-6 to obtain primary seed culture.

[0095] Secondary seed culture: The above primary seed culture was transferred to fresh BMG liquid medium at an inoculation rate of 1% (v / v) and cultured with shaking at 30℃ and 220 rpm until the OD reached 10-12 to obtain the secondary seed culture.

[0096] Fermentation tank culture

[0097] Fermentation system construction: FM22 fermentation medium was used as the basal medium. The above secondary seed liquid was inoculated into a 5L fermenter at an inoculation rate of 5% (v / v). The fermentation substrate was 3L.

[0098] Initial culture stage: The initial culture temperature was set at 30℃, the stirring speed at 300 rpm, and the aeration rate at 2 vvm (volume of air / volume of culture medium / min); the pH of the fermentation broth was adjusted by automatically adding ammonia water to maintain it at 5.2±0.1; a dissolved oxygen feedback control strategy was adopted to maintain the dissolved oxygen (DO) concentration in the fermentation broth above 25% by adjusting the stirring speed.

[0099] Glycerol feeding stage: After about 18 hours of initial culture, when the glycerol in the FM22 medium is completely consumed (DO value rises rapidly to above 70%), glycerol feeding is started. Dissolved oxygen feedback control is used. When the DO value is higher than 25%, glycerol is fed at a rate of 1.0 mL / min until the wet cell concentration in the fermentation broth reaches 200 g / L, at which point glycerol feeding is stopped.

[0100] Methanol induction phase: After glycerol feeding, the stirring speed was increased to 900 rpm, the temperature was lowered to 25℃, and methanol feeding was started to induce expression. During the first 4 hours of induction, the methanol feed rate was increased to 0.05 mL / min to promote cell adaptation to the change in carbon source from glycerol to methanol. After 4 hours of induction, the methanol feed rate was adjusted to 0.21 mL / min, and the dissolved oxygen feedback control strategy was still used (feeding was initiated when DO value ≥ 25%). Induction culture was continued for 96 hours, after which fermentation was terminated.

[0101] Samples were taken every 24 hours during fermentation, and the supernatant from the collected samples was analyzed by SDS-PAGE protein electrophoresis. The results are as follows: Figure 3 As shown, a distinct single protein band appears at a position of approximately 35 kDa, indicating successful fermentation expression of recombinant type XVII collagen COL1510 and TAT-COL1510. Furthermore, COL1510 shows a degradation band below the target band, while TAT-COL1510 shows no degradation band, indicating that the transmembrane peptide TAT has a positive effect on protein stability.

[0102] Example 6: Purification of recombinant collagen COL1510 and recombinant collagen TAT-COL1510 containing membrane-penetrating peptides

[0103] In this embodiment, the target protein in the fermentation broth obtained in Example 5 was separated and purified by combining ammonium sulfate precipitation with dialysis and freeze-drying.

[0104] The fermentation broth obtained in Example 5 was filtered through a plate and frame filter to remove the bacterial cells, and the supernatant was collected. The supernatant was then filtered through a hollow fiber membrane with a pore size of 0.45 μm to remove residual bacterial fragments and macromolecular impurities, and the permeate was collected. The permeate was then filtered through a membrane with a pore size of 10 kDa to concentrate and enrich the target protein, and the concentrate was collected.

[0105] Set up ammonium sulfate concentration gradients of 20%, 30%, 40%, 50%, 60%, and 70% to precipitate the target protein, and calculate the amount of ammonium sulfate to be used according to the formula (m=76.7×V×target saturation÷100).

[0106] m represents the required mass of ammonium sulfate in grams; 76.7 represents the solubility of ammonium sulfate in water at 25°C, meaning 100g of water can dissolve 76.7g of ammonium sulfate; V represents the volume of the protein solution in mL.

[0107] Slowly add the required mass of ammonium sulfate solid while stirring, ensuring it dissolves completely. Do not add the ammonium sulfate too quickly. Let the solution stand at 4°C for 4 hours to allow the protein to precipitate fully from the ammonium sulfate. Centrifuge the solution after standing, typically at 12000 rpm for 15 minutes at 4°C. Collect the precipitate and discard the supernatant. Dissolve the precipitate in deionized water, stirring slowly to avoid foaming the protein solution.

[0108] The precipitates and supernatants obtained under various ammonium sulfate concentration gradients were analyzed by SDS-PAGE protein electrophoresis, and the results are as follows: Figure 4 and Figure 5As shown, a 30% ammonium sulfate concentration exhibited good separation and purification effects on recombinant type XVII collagen COL1510 and TAT-COL1510. Therefore, the membrane-encapsulated concentrates of the above two collagens were treated with a 30% ammonium sulfate concentration, and the resulting precipitates were dissolved in deionized water.

[0109] Ultrafiltration desalination: The above protein solution is filtered through a membrane with a pore size of 10 kDa to remove salt, and the concentrate is collected.

[0110] Freeze-drying: Transfer the desalted protein solution to a freeze-drying bottle, place it in a freeze dryer, and freeze-dry according to the preset freeze-drying curve (pre-freezing temperature -40℃, pre-freezing time 4 h; sublimation drying temperature -20℃, vacuum degree 10 Pa, time 24 h; desorption drying temperature 25℃, vacuum degree 5 Pa, time 8 h) to obtain collagen freeze-dried powder, which is then sealed and stored at -20℃ for later use.

[0111] Purity testing

[0112] The purity of the above lyophilized powder was determined by high performance liquid chromatography (HPLC), under the following specific conditions:

[0113] Column: BioCore SEC-150 gel filtration column;

[0114] Mobile phase: 50 mmol / L phosphate buffer (pH 6.8) containing 300 mmol / L NaCl;

[0115] Elution method: isocratic elution;

[0116] Flow rate: 0.5 mL / min;

[0117] Injection volume: 5 μL;

[0118] Column temperature: 30℃;

[0119] Detection wavelength: 220 nm;

[0120] Sample preparation: Dilute the lyophilized powder to 1 mg / mL with the mobile phase, filter through a 0.22 μm filter membrane, and then load the sample.

[0121] Test results: The purity of TAT-COL1510 collagen freeze-dried powder is 93.83%, and the purity of COL1510 collagen freeze-dried powder is 97.39%, which meet the purity requirements for subsequent applications.

[0122] Example 7 Transdermal Performance Test of Recombinant Collagen

[0123] This embodiment aims to quantitatively evaluate the transdermal penetration amount and rate of recombinant human collagen TAT-COL1510 and COL1510 using an in vitro transdermal model, clarify their transdermal capabilities in a simulated skin environment, and provide data support for the application of this recombinant collagen in skin care products, medical dressings and other fields.

[0124] TAT-COL1510 and COL1510 lyophilized products were dissolved in deionized water to prepare 1.0% collagen solutions. The TAT-COL1510 collagen solution was labeled as the experimental group, and the COL1510 collagen solution was labeled as control group 1. A blank matrix solution without collagen (containing 5% glycerol and 0.1% preservative) was labeled as control group 2. Three replicates were set up for each group to ensure experimental repeatability.

[0125] Fresh pig ear skin was selected, subcutaneous fat and connective tissue were removed, and the skin was washed with physiological saline until no bloodstains were found. The skin was then cut into circular pieces with a diameter of 3 cm and stored frozen at -20℃. Before use, the skin was thawed at room temperature and fixed with a polytetrafluoroethylene membrane for later use. (The skin pieces were tested and found to have good barrier integrity and the transdermal permeation of the blank matrix was below the detection limit.)

[0126] Pig ear skin was fixed between the supply and receiving chambers of the Franz diffusion cell, with the stratum corneum facing the supply chamber and the dermis facing the receiving chamber, ensuring a tight fit between the skin and the cell wall without any air bubbles. Preheated receiving solution to 32°C was poured into the receiving chamber, the liquid level covering the dermis of the skin. A constant temperature water bath was started to maintain the system temperature at 32±0.1°C (simulating the surface temperature of human skin), and a magnetic stirrer was used to continuously stir at 500 rpm. After equilibration for 30 minutes, 0.5 mL of experimental and control samples were added to the supply chamber, and the supply chamber was sealed to prevent solvent evaporation. At sampling time points of 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, 0.5 mL of receiving solution was drawn from the receiving chamber, and an equal amount of preheated fresh receiving solution was added simultaneously to ensure a constant volume in the receiving chamber.

[0127] Transdermal transdermal absorption rate (TVR): The concentration of collagen in the receiving solution was determined using the BCA protein quantification method. 50 μL of the receiving solution sample was taken at each time point, and 200 μL of a mixture of reagents A and B was added according to the BCA kit instructions. After incubation at 37°C for 30 min, the absorbance was measured at 562 nm using a UV-Vis spectrophotometer. The concentration of collagen in the receiving solution was calculated based on a pre-plotted collagen standard curve (R² = 0.998), and the TVR and cumulative TVR (Qn) were calculated using the following formulas:

[0128] Q = C×V (Equation 1), where C is the collagen concentration in the receiving fluid (μg / mL) and V is the volume of the receiving chamber (mL).

[0129] Qn = Q + Σ(Qi×Vi / Vs) (Equation 2), where Qi is the transdermal volume before the i-th sampling, Vi is the volume of the i-th sampling, and Vs is the volume of the receiving chamber.

[0130] A transdermal permeation curve was plotted with cumulative transdermal volume (μg / cm²) on the ordinate and time (h) on the abscissa. The linear portion of the curve (usually 2-8 h) was selected to calculate the permeation rate (Jss), i.e., the slope of the linear regression equation (μg / (cm²·h)). Simultaneously, the cumulative transdermal volume (Q24) and transdermal permeability (transdermal permeability = Q24 / total collagen mass in the supply chamber × 100%) were calculated. Data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 22.0 software, with P < 0.05 considered statistically significant. The transdermal performance indicators of collagen in each group are shown in Table 1 below.

[0131] Table 1. Transdermal performance indicators of collagen in each group

[0132]

[0133] These results indicate that, compared with collagen COL1510 without the addition of transdermal peptides, transdermal peptide-fused collagen TAT-COL1510 has significantly enhanced transdermal penetration capability and can be used as an effective collagen raw material for transdermal delivery of active ingredients or for skin repair.

[0134] Example 8: Experiment on the promotion of cell proliferation by recombinant collagen

[0135] This embodiment aims to evaluate the effect of collagen on the proliferative activity of human dermal papilla cells (hDPCs) and verify its potential application in promoting hair growth.

[0136] Human dermal papilla cells (hDPCs) were purchased from ScienCell Research Laboratories (or ATCC), and were passage 3-5 cells. They were cultured in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. Collagen TAT-COL1510 and COL1510 were prepared as 1 mg / mL stock solutions, sterilized by filtration through a 0.22 μm filter, and diluted to the required concentration (100 μg / mL) before use. The blank control group contained only culture medium and cells. The experimental groups were supplemented with 100 μg / mL collagen, with three replicates per group, and cultured for 48 hours. Cell proliferation was detected using the CCK-8 assay (Cell Counting Kit-8). 10 μL of CCK-8 reagent was added to each well, and after incubation for 2 hours, the absorbance (OD value) at 450 nm was measured using a microplate reader. Compared with the blank control group, the experimental group supplemented with collagen showed a significant cell proliferation-promoting effect. The results are shown in Table 2 below (in terms of OD). 450 express):

[0137] Table 2 Results of recombinant collagen promoting cell proliferation

[0138]

[0139] These results indicate that both collagen TAT-COL1510 and COL1510 have the potential to promote the activity of hair follicle-associated cells (HACs), with TAT-COL1510 demonstrating a stronger ability to do so, thus supporting its application in promoting hair growth or skin repair.

[0140] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

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

7.

2. A polynucleotide, comprising, The polynucleotide encodes the recombinant collagen XVII according to claim 1.

3. The polynucleotide of claim 2, wherein, The nucleotide sequence of the polynucleotide is shown as SEQ ID NO.

8.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide according to claim 2 or 3.

5. A host cell, characterized in that, The host cell contains the recombinant expression vector according to claim 4 or has the polynucleotide according to claim 2 or 3 chromosomally integrated or expresses the recombinant collagen XVII according to claim 1.

6. A method of producing the recombinant collagen XVII according to claim 1, wherein Comprising: culturing the host cell according to claim 5 so as to express the recombinant collagen XVII; and isolating and purifying the recombinant collagen XVII.

7. Use of the recombinant collagen XVII of claim 1 in the preparation of a cosmetic or medical aesthetic product; wherein, The cosmetic is a skin care product.

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