Stable recombinant XVII type collagen and application thereof

Abstract

The invention relates to the technical field of genetic engineering, in particular to stable recombinant XVII type collagen and application thereof. The stable recombinant XVII type collagen has a stable molecular structure, the molecular weight of the stable recombinant XVII type collagen is smaller than that of a complete sequence, and the stable recombinant XVII type collagen contains functional active sites and can give consideration to transdermal absorption and biological structure stability; the stable recombinant XVII type collagen can realize industrial production, and has good application in the fields of medicines, medical equipment, biological materials, tissue engineering products, cosmetics or health care products and the like.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a stable recombinant type XVII collagen and its applications. Background Technology

[0002] Collagen, a natural protein macromolecule, is an important biodegradable material. In the human body, collagen exhibits low immunogenicity, biodegradability, biocompatibility, and the ability to promote cell growth and hemostasis. Due to these properties, the synthesis and preparation of recombinant collagen, the design and development of active collagen peptides, the exploration of novel functions of collagen-based materials, and the development and manufacturing of various types of collagen-based materials and medical materials have become current research hotspots. Currently, some tissue engineering materials and biomedical materials made using collagen have achieved clinical success and are widely accepted by clinicians and patients. A 2021 study by Mol Clin Oncol et al. indicated that Col17a1 in epidermal stem cells plays a crucial role in the development of melanoma; a 2024 study by J Inflamm Res. et al. showed that rhCol 17 collagen spray can effectively treat oral ulcers, exhibiting anti-inflammatory and tissue-healing-accelerating effects; Mol Med Rep reported that RHCXVII maintains basement membrane integrity by increasing the expression levels of type I, type IV, and laminin subunits β3 and ITGA6 in the ECM and inhibiting protein phosphorylation in the MAPK and Wnt pathways. Most of the expression vectors mentioned in these studies are mammalian cells and are primarily research-oriented. Patent CN118344464 mentions the expression of recombinant XVII type collagen peptide tandem repeats, with the expression material showing good efficacy in hemostatic materials. Patent CN118702825 describes the fusion of recombinant XVII type collagen with epidermal growth factor receptor (EGFR) expression in epidermal cells, aiming to achieve better skin self-repair capabilities.

[0003] Type XVII collagen (COL17) is a transmembrane protein located in the epidermal basement membrane region. It is a key component connecting the cytoskeleton and the basement membrane, mediating skin homeostasis and playing a crucial role in maintaining skin integrity. Traditional animal-derived collagen requires large-scale livestock farming to supply raw materials, leading to significant consumption of land, water, and food resources. Furthermore, type XVII collagen is present in low amounts in tissues and can only be obtained through recombinant methods. Current technologies utilize gene recombination to obtain recombinant type XVII collagen, preserving the original humanized sequence, exhibiting low immunogenicity, and a short growth cycle. However, it is primarily dependent on the host system, with expression systems mainly including prokaryotic, eukaryotic, and mammalian expression systems. E. coli expression (a prokaryotic expression system) is low-cost and short-cycle, but it cannot achieve post-translational modification of exogenous proteins and is prone to generating pyrogens. Pichia pastoris expression (a eukaryotic expression system) can complete basic post-translational modification, secretory expression, reduce fermentation and purification costs, has high yield, and does not generate pyrogens. This patent utilizes the Pichia pastoris expression system to achieve recombinant expression of recombinant type XVII collagen. The developed recombinant type XVII collagen has low immunogenicity, good hydrophilicity, and high stability, and has good application prospects in subsequent pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics, or health products. Summary of the Invention

[0004] The purpose of this invention is to provide a stable recombinant type XVII collagen and its applications. This invention uses genetic engineering techniques to design the amino acid sequence of fully human type XVII collagen and expresses the exogenous protein using Pichia pastoris as an expression vector to obtain a stable recombinant type XVII collagen with good stability, high water solubility, and low immunogenicity. It has excellent applications in the fields of pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics, and health products.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a stable recombinant type XVII collagen, the stable recombinant type XVII collagen comprising: a polypeptide with an amino acid sequence as shown in SEQ ID NO. 6; or, a polypeptide with an amino acid sequence as shown in SEQ ID NO. 7.

[0006] The present invention also provides a gene encoding the stable recombinant type XVII collagen, the nucleotide sequence of which is shown in any one of SEQ ID NO. 8-9.

[0007] The present invention also provides an expression vector comprising a gene encoding the stable recombinant type XVII collagen and an empty vector.

[0008] Preferably, the empty vector is a pPIC9K vector or a pPICZαA vector.

[0009] The present invention also provides a genetically engineered bacterium comprising the aforementioned expression vector and an empty host bacterium.

[0010] Preferably, the empty host bacterium is a eukaryotic cell.

[0011] Preferably, the host cell is Pichia pastoris. More preferably, the recombinant engineered strain is Pichia pastoris GS115-pPIC9K-17A6 ( Komagataella phaffi ), deposited on December 8, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39094; or Pichia pastoris GS115-pPIC9K-17A3 ( Komagataella phaffi The strain was deposited on December 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 39095.

[0012] It is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCC No. 39095 (GS115pPIC9K-17A3) and CGMCC No. 39094 (GS115pPIC9K-17A6).

[0013] The present invention also provides a method for preparing the genetically engineered bacteria, comprising the following steps: The expression vector was linearized by enzyme digestion and transformed into Pichia pastoris. The Pichia pastoris genetically engineered strain expressing stable recombinant type XVII collagen was obtained by screening through the G418 resistance gradient.

[0014] The present invention also provides the application of the stable recombinant type XVII collagen, or the gene encoding the stable recombinant type XVII collagen, or the expression vector containing the gene, or the genetically engineered bacteria containing the expression vector, in the preparation of pharmaceuticals, cosmetics, health products, or medical devices.

[0015] The present invention has the following technical effects and advantages: This invention utilizes synthetic biology, genetic engineering, and biotechnology to design the original amino acid sequence of human type XVII collagen, removing MMP restriction sites, N-glycosylation sites, and unstable triplets. Peptides containing active sites that are easily expressed are selected. This designed, smaller molecular weight amino acid sequence allows for the in vitro construction of recombinant collagen with good stability while meeting functional requirements. The Pichia pastoris expression system does not generate pyrogens, allows for high-density fermentation with high expression levels, has a short fermentation cycle, and low cost. Secretory expression facilitates subsequent purification. The stable recombinant type XVII collagen of this invention has a stable molecular structure, a smaller molecular weight than the full sequence ratio, and contains functional active sites, which can achieve both transdermal absorption and biological structural stability. The stable recombinant type XVII collagen can be industrialized and has good application prospects in the fields of pharmaceuticals, medical devices, biomaterials, tissue engineering products, cosmetics or health products. Attached Figure Description

[0016] Figure 1 The images show the enzyme digestion identification results of the recombinant expression vector in Example 1; the left side of the image shows the recombinant expression vector pPIC9K-17A3, and the right side shows the recombinant expression vector pPIC9K-17A6.

[0017] Figure 2 The results of agarose gel electrophoresis of the target protein encoding gene in Example 1 are shown; the left side of the figure shows stable recombinant type XVII collagen 17A3, and the right side shows stable recombinant type XVII collagen 17A6.

[0018] Figure 3 The results of SDS-PAGE protein electrophoresis of the target protein in Example 1 show that the molecular size is between 17-40KD; the left side of the figure shows stable recombinant type XVII collagen 17A3, and the right side shows stable recombinant type XVII collagen 17A6.

[0019] Figure 4 The mass spectrometry identification results of the target protein in Example 1 are shown; a is stable recombinant type XVII collagen 17A3, b is stable recombinant type XVII collagen 17A6, and the red line represents the sequence for comparison.

[0020] Figure 5 The results show the expression identification of the target protein after high-density fermentation in Example 1; the left side of the figure shows stable recombinant type XVII collagen 17A3, and the right side shows stable recombinant type XVII collagen 17A6.

[0021] Figure 6The results are SDS-PAGE verification results of the purified target protein in Example 1; the left side of the figure shows stable recombinant type XVII collagen 17A3, and the right side shows stable recombinant type XVII collagen 17A6.

[0022] Figure 7 The cell adhesion results of each stable recombinant type XVII collagen in Example 2 are shown; where a and b are stable recombinant type XVII collagen 17A6, and c and d are stable recombinant type XVII collagen 17A3.

[0023] Figure 8 The cell migration results of each stable recombinant type XVII collagen in Example 3 are shown; where a and b are stable recombinant type XVII collagen 17A6, and c and d are stable recombinant type XVII collagen 17A3.

[0024] Figure 9 Cellular activity of each stable recombinant type XVII collagen in Example 4; where a is stable recombinant type XVII collagen 17A6 and b is stable recombinant type XVII collagen 17A3.

[0025] Figure 10 The cell stability of each stable recombinant type XVII collagen under different conditions in Example 5 is shown in Figure 5. Among them, a is the SDS-PAGE detection result of stable recombinant type XVII collagen 17A6 and 17A3 under repeated freeze-thaw conditions, b is the SDS-PAGE detection result of stable recombinant type XVII collagen 17A6 and 17A3 under different pH conditions, and c is the SDS-PAGE detection result of stable recombinant type XVII collagen 17A6 and 17A3 at different temperatures. Detailed Implementation

[0026] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] This invention uses the fully human type XVII collagen sequence as a reference, removing unstable triplets, MMP cleavage sites, and N-glycosylation sites. Fragments with good hydrophilicity are selected, with an isoelectric point greater than 7 and a molecular weight between 15 kDa and 50 kDa. Active site prediction and immunogenicity prediction are performed on the selected fragments (https: / / www.iedb.org). The target protein is expressed using Pichia pastoris as an expression system, with secretory expression to increase purification efficiency.

[0028] Example 1 1. Sequence design of stable recombinant type XVII collagen COL17A1 is primarily expressed in skin epithelial cells and mainly located within basement membrane junctions. It plays a crucial role in the adhesion between the skin and dermis. Therefore, a low-molecular-weight, highly biocompatible recombinant humanized COL17A1 protein (rhCOL17) shows promising application prospects in biomaterials and medical devices.

[0029] Sequences containing integrin binding sites "KGD, GEKGER" were selected, avoiding triads with a thermostability score of "0", such as: GPI, GIA, GDK, GAA, GEA, GAK, GAD, GPI, GET, and GPF. N-glycosylation sites (NXS / T) were also avoided. Based on these design principles, sequence selection was performed, and the selected sequences were spliced ​​and tandem to form fragments of approximately 15-50 kDa for expression, yielding stable recombinant type XVII collagen with good stability and water solubility.

[0030] Using the sp Q9UMD9 COHA1_HUMAN Collagen alpHa-1(XVII) chain sequence in the uniprot sequence as a reference, the following segments were selected: helical region 15 (597-646, 50 AA), helical region 15 (680-736, 57 AA), helical region 10 (951-977, 27 AA), helical region 4 (1215-1235, 21 AA), and helical region 1456-1482 (27 AA). These are represented as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. The specific amino acid sequences are as follows: SEQ ID NO.1~5: SEQ ID NO.1: GHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPG SGEKGER; SEQ ID NO.2: GPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEP; SEQ ID NO.3:GPPGPPGPQGPKGDKGDPGVPGALGIP; SEQ ID NO.4:GPPGPPGPPGPRGPPGVSGAL; SEQ ID NO.5: GPAGPPGHPGPPGPRGHKGEKGDKGDQ; Fragments SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 were concatenated and repeated twice to obtain the target sequence 17A3. This target sequence was inserted into the pPIC9K vector between the EcoRI and NotI restriction sites after the alpha-factor secretion signal. The amino acid "LEKR" was added before the target sequence to facilitate intracellular Kex2 digestion after expression, yielding the native N-terminal target sequence. A terminator was added to the end of the target sequence to obtain sequence SEQ ID NO.6. The expressed target protein 17A3 is the amino acid sequence following the double slashes of "LEKR".

[0031] SEQ ID NO.6: LEKR / / GHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGPPGPPGPQGPKGDKGDPGVPGALGIPGPPGPPGPPGPRGPPGVSGALGPAGPPGHPGPPGPRGHKGEKGDKG DQGHPGPQGPKGQKGSVGDPGMEGPMMGQRGREGPMGPRGEAGPPGSGEKGERGPVGLQGLRGEVGLPGVKGDKGPMGPPGPPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGPPGPPGPQGPKGDKGDPGVPGALGIPGPPGPPGPPGPRGPPGVSGALGPAGPPGHPGPPGPRGHKGEKGDKGDQ*. Fragment SEQ ID NO.5 was repeated 7 times to obtain the target sequence 17A6. The target sequence was inserted into the pPIC9K vector between the EcoRI and NotI restriction sites after the alpha-factor secretion signal. The amino acid "LEKR" was added before the target sequence to facilitate intracellular Kex2 digestion after expression, yielding the natural N-terminal target sequence. A terminator was added to the end of the target sequence to obtain sequence SEQ ID NO.7. The expressed target protein 17A6 is the amino acid sequence after the double slashes of "LEKR".

[0032] SEQ ID NO.7: LEKR / / GPAGPPGHPGPPGPRGHKGEKGDKGDQGPAGPPGHPGPPGPRGHKGEKGDKGDQGPAGPPGHPGPPGPRGHKGEKGDKGDQGPAGPPGHPGP PGPRGHKGEKGDKGDQGPAGPPGHPGPPGPRGHKGEKGDKGDQGPAGPPGHPGPPGPRGHKGEKGDKGDQGPAGPPGHPGPPGPRGHKGEKGDKGDQ*. 2. Constructing recombinant expression vectors DNA encoding the amino acid sequences SEQ ID NO. 6 and SEQ ID NO. 7 was synthesized, with corresponding nucleic acid sequences SEQ ID NO. 8 and SEQ ID NO. 9. These nucleic acid sequences were constructed into the pPIC9K vector, and an insertion was made between the EcoRI and NotI restriction sites after the alpha-factor sequencing signal. The N-terminal "LEKR" amino acid sequence was digested with Kex2 enzyme by Pichia pastoris after expression to obtain the natural N-terminal target sequence protein. The constructed recombinant expression vectors were named pPIC9K-17A3 and pPIC9K-17A6. The nucleic acid sequences are shown below, with underlined EcoRI and NotI restriction sites representing the sites. The expression vectors were purchased from GenScript, and the target sequences underwent codon optimization to suit Pichia pastoris expression.

[0033] SEQ ID NO. 8: gaattc gcggccgc; SEQ ID NO. 9: Gaattcc ttgagaagcgtggacctgctgggccaccaggacaccaggccccccaggcccgcgaggacacaagggtgaaaagggggacaagggtgatcagggaccagccggccccccgggacatccgggtccgcccggaccgaggggtcacaa aggcgagaaaggcgacaaaggtgaccaaggacccgcaggtcctccaggacaccctgggccgccgggaccgcgggggcataaaggggaaaaaggcgataaaggcgatcagggtccagctggccctcctgggcaccctgggcccccc ggcccgagaggtcataagggagaaaaaggggacaagggtgaccaagggccggcgggccctcctgggcatcctggcccaccgggaccccgcggccataaaggtgaaaaaggtgataagggcgatcaggggcccgcagggcctccgg gtcatccaggtccgccgggcccaaggggccacaagggagagaagggagataaaggtgatcaagggccagcgggtcctcctgggcaccccggcccacccggaccccgaggtcataaaggagagaagggggacaagggtgatcaatga gcggccgc; 3. Constructing recombinant collagen engineered strains Expression vectors pPIC9K-17A3 and pPIC9K-17A6 were linearized using Sal I rapid digestion enzyme (purchased from TaKaRa, Dalian; specific procedures followed the reagent instructions) at 37℃. 10 μg of plasmid was then digested. The linearized plasmid was collected and purified using a PCR product purification kit (purchased from Sangon Biotech, Shanghai; specific procedures followed the kit instructions). The enzyme digestion identification results are as follows. Figure 1 As shown in the image, the left side represents undigested plasmids, and the right side represents linearized plasmids digested with enzymes. The plasmids exist in three states: supercoiled, open-circular, and linear. Their migration rates in the nucleic acid electrophoresis gel decrease sequentially. The experimental results are shown below. Figure 1As shown, the linearized plasmid has a slower migration rate, proving that the plasmid is completely linearized. The linearized plasmid has a slower migration rate than the plasmid in the non-linearized supercoiled state.

[0034] Competent cells of strain GS115 (purchased from Thermo Fisher Scientific, C18100) were prepared and dispensed in 40 μl tubes for subsequent electroporation. The linearized plasmid was electroporated into GS115 Pichia pastoris competent cells at a nucleic acid content of 1-5 μg, voltage 1.5 kV, and time 5.5 ms. 200 μL of the electroporated bacterial culture was plated onto MD plates and incubated upside down at 30°C for 2-5 days for auxotrophic selection until a single colony (His...) was identified. + (Positive bacteria growth) appeared. After auxotrophic screening, the bacterial cells were washed off MD plates with sterile water and cultured at 5 × 10⁻⁶. 6 The bacteria were plated on YPDG (500 μg / ml, 1 mg / ml, 2 mg / ml, "G" represents genetic mycotoxin, G418) plates and incubated upside down at 30°C for 2-5 days until single colonies appeared. Plates with higher concentrations of antibiotics showed higher integrated copy numbers and higher expression levels. The obtained transformants were named GS115 / pPIC9K-17A3 and GS115 / pPIC9K-17A6.

[0035] 4. Validation of target gene transfer After YPDG resistance plate screening, Pichia pastoris colony PCR verification was performed using polymerase chain reaction (PCR). 200 μl of BMD1 medium was added to 96-well plates containing strains GS115 / pPIC9K-17A3 and GS115 / pPIC9K-17A6. Single colonies from each group were selected using sterile toothpicks and inoculated into the 96-well plates. The plates were incubated at 30°C for 24 h. 80 μl of bacterial culture was extracted, and the template was extracted using the alkaline lysis method. The plates were centrifuged for 5 min, and the supernatant was discarded. 80 μl of sterile water was added, the bacterial cells were resuspended, and the plates were centrifuged for 5 min, and the supernatant was discarded. 10 μL of 0.02 mol / L NaOH solution was added, and the bacterial cells were vortexed for resuspending. The prepared bacterial culture was then placed on a PCR instrument, boiled at 99°C for 10 min, and then allowed to cool to room temperature before use. Centrifuge for 10 min using a handheld centrifuge, and take 2 μL of the supernatant as a template. rTaq (purchased from TarKa) is used as the PCR polymerase, and the primers are shown in Table 1 below: Table 1: Primer information for PCR validation

[0036] Reaction conditions: 94℃, 4 min; 98℃, 10 s; 55℃, 30 s; 72℃, 140 s; 30 cycles, 72℃, 5 min. Polymerase chain reaction was performed under the above reaction system and conditions, and the target band was verified by nucleic acid electrophoresis. Experimental results are as follows: Figure 2As shown in Table 1, the size of the target band is shown in the PCR results, which confirm that the plasmid was successfully integrated into the Pichia pastoris genome.

[0037] 5. Induced expression of recombinant engineered strains The GS115 / pPIC9K-17A3 and GS115 / pPIC9K-17A6 engineered bacteria, whose colony PCR verification was correct, were placed in 100mL Erlenmeyer flasks containing 15mL of BMGY or YPD medium and incubated at 28-30℃ and 220rpm for 18-24h until the OD600 reached 6-10. The cells were then centrifuged at 1500-3000g for 5min at room temperature, collected, and resuspended in BMMY medium to approximately OD600 10. The cells were then placed on a shaker at 28-30℃ and 220rpm for 3 days of continued growth, with 100% methanol added to the medium every 24h until a final concentration of 1% was reached. After methanol induction, the expression supernatant was collected and verified by SDS-PAGE protein electrophoresis using a Future PAGE 12% protein electrophoresis gel (purchased from Boyi Biotechnology Co., Ltd.). The experimental results are as follows: Figure 3 As shown, the theoretical size of the 17A3 target protein is 34168.20 Da, and the theoretical size of the 17A6 target protein is 17985.46 Da. Gray-scale analysis of the experimental results indicates a purity of over 85%, and the protein is secreted into the supernatant. SDS-PAGE results show that the apparent molecular weight is slightly larger than the theoretical molecular weight, which is related to the amino acid sequence and structure of the recombinant collagen, resulting in a slower migration rate compared to normal proteins.

[0038] 6. Protein expression identification Protein electrophoresis was performed on GS115 / pPIC9K-17A3 and GS115 / pPIC9K-17A6 using a Future PAGE 12% precast protein electrophoresis gel (purchased from Boyi Biotechnology Co., Ltd.). Electrophoresis was stopped when the proteins migrated to the bottom of the gel. After staining and destaining, the size of the protein bands matched the expected apparent migration size. The target bands were cut off and digested with trypsin. Nano-HPLC-MS / MS mass spectrometry was used to detect the trypsin-digested peptides of recombinant collagen (the detection was performed by Suzhou Putai Biotechnology Co., Ltd.). The trypsin cleavage sites are K and R. The digested peptides generally end with K or R. During database retrieval and software analysis, the peptide segment between K and R was selected as the detection peptide. The detected peptides were compared with the theoretical sequence. Mass spectrometry analysis showed that the similarity to the theoretical amino acid sequence of collagen was over 85%. For example, the results are as follows. Figure 4As shown, a is 17A3, and b is 17A6. The red lines represent the sequences that were compared. If the N-terminal sequence of a sequence is the middle part in the database sequence, and the theoretical enzyme-digested peptide used in the software analysis has one more amino acid than the one in the database, then the sequence cannot be aligned. However, it can be compared with the theoretical sequence. Identification shows that the target bands of GS115 / pPIC9K-17A3 and GS115 / pPIC9K-17A6 can be expressed in extracellular secretion, and the mass spectrometry sequence alignment is consistent with the theoretical sequence.

[0039] 7. High-density fermentation, culture, and protein purification (1) High-density fermentation Culture media and components used in high-density fermentation processes Seed culture medium (YPG): yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L; Fermentation medium: NH4H2PO4 190.4 g / L, KH2PO4 10.06 g / L, CaSO4•2H2O 1.18 g / L, K2SO4 18.2 g / L, MgSO4•7H2O 14.9 g / L, glycerol 40 g / L; After high-temperature sterilization, PTM1 was added after the fermentation medium cooled to room temperature, and the pH was adjusted to 5.0 with ammonia. Feeding medium: 50% w / v glycerol, with 12 mL PTM1 micronutrients per liter; Induction medium: 100% methanol, with 12 mL PTM1 micronutrients per liter; PTM1: sterilized by filtration through a 0.22 μm filter membrane and stored at 4℃.

[0040] Fermentation was carried out using a 5L glass jar fermentation system. One frozen sample of the target strain was thawed and inoculated into a sterilized shake flask culture medium at 180 μL (0.24% inoculum). The shaker was pre-started and programmed at 220 rpm and 30°C. The inoculated shake flask was then placed in the shaker. OD600 was measured based on the growth of the shake flasks until it reached a range of 4-8, at which point the shake flask culture was considered complete. The fermentation medium was then transferred to a 5L fermenter. 1 ml of defoamer was added, the fermenter was assembled and sealed, and sterilized at 121°C for 20 minutes. After the fermenter cooled naturally, the necessary piping was installed. 150 ml of the glycerol supplementation solution was added to a 2L feed bottle. The feed bottle was assembled and sealed, and sterilized at 121°C for 20 minutes. After cooling, PTM1 was aseptically added to the glycerol feed bottle in a laminar flow hood. Measure 1500ml of methanol into a sterilized 5L feed bottle. In a laminar flow hood, aseptically add 18ml of PTM1 to the methanol feed bottle. Measure 300-500ml of ammonia into a 500mL sterilized feed bottle for later use. Before inoculation, pre-set the tank temperature to 30℃, pH 5.0, aeration to 6L / min, stirring to 600rpm, and tank pressure to 0.05MPa. Calibrate dissolved oxygen to 100%. After calibration, reduce the aeration rate to 2.0-4.0L / min and the stirring to 300rpm. Sample the seed culture from the shake flask and test its OD600 and microscopic indicators. If the OD600 is within the range of 4-8, the shake flask culture is complete and ready for transfer. Transfer 6530ul of PTM1 to a sterile centrifuge tube and aseptically add it along with 150ml of seed culture at the inoculation port under flame protection (inoculation volume 10%).

[0041] Basic fermentation stage: Initial fermentation conditions in the fermenter: temperature controlled at 30℃, aeration rate of 2.0L / min, pH maintained at 5.0, stirring at 300-900rpm, tank pressure of 0.05Mpa, dissolved oxygen >30%. When dissolved oxygen drops below 30%, increase the stirring speed to ensure dissolved oxygen remains above 30%. After the glycerol in the substrate is depleted and dissolved oxygen rebounds, the basic fermentation stage ends, and the glycerol supplementation stage begins.

[0042] Fermentation stage of supplementation: Keep the culture conditions unchanged, and add supplementation at an actual feeding rate of 25-26 ml / h. During the feeding process, increase the aeration rate and stirring speed to ensure that the dissolved oxygen is above 20%. After the supplementation is completed, stop adding supplementation. Start timing 30 minutes from the beginning of dissolved oxygen rebound. The strain is then starved, the supplementation stage ends, and the induction stage begins.

[0043] Fermentation induction phase: pH was maintained at 6.0, temperature at 26.5℃, tank pressure at 0.04-0.06 MPa, and dissolved oxygen at 20-30%. Feeding rate, stirring, tank pressure, and aeration were adjusted according to the growth and expression status to ensure dissolved oxygen was maintained above 20% during the induction phase. High-density fermentation protein expression identification results are as follows: Figure 5 As shown, the target protein is normally secreted and expressed in the extracellular supernatant.

[0044] (2) Protein purification The culture medium and components used in the protein purification process were: MMC large scale, 2.6×22cm; the purification equipment was: a Seppu protein chromatography system with high performance liquid chromatography; sample preparation was: pH adjusted to 4.5 with dilute HAc, conductivity below 8mS / cm; the loading volume was 30mg / ml. Buffer A: 50mM HAc-NaAc, pH 4.5; Buffer B: 50mM HAc-NaAc, 1M NaCl, pH 4.5; CIP: 0.5M NaOH / 1M NaOH; Preservative solution: 20% ethanol.

[0045] Experimental Procedure: The flow rate throughout the chromatography was 14.5 ml / min (retention time 8 min). The flow rates for pretreatment and post-CIP could be appropriately reduced. Pre-CIP: Rinse column preservative with purified water 3 CV, then rinse with 0.5 M NaOH for at least 3 CV for at least 30 min; Equilibration: Rinse with buffer B for 3 CV, then rinse with buffer A for 5 CV until conductivity and pH are stable and consistent with buffer A; Sample Loading: Load sample at a capacity of 30 mg / ml, sample pH 4.52, conductivity 5.47 mS / cm; Washing: Rinse with buffer A for 5 CV; Impurity Washing: Wash with 20% B for 5 CV, then wash with 25% B for 2 CV; Elution: Elute with 100% B until A reaches 220 nm and drops to 200 mAU, then stop collecting; CIP: Rinse with 0.5M NaOH for at least 3 CV for at least 30 min (it is recommended to replace with 1M NaOH CIP once every 3 uses, 3 CV); Equilibration (repeat if purifying again): Rinse with buffer B for 3 CV, then rinse with buffer A for 5 CV until conductivity and pH are stable and consistent with buffer A; Storage: Rinse with purified water for at least 5 CV, then rinse with 20% ethanol for 3 CV and store. Collect purified samples for SDS-PAGE verification. Experimental results are as follows: Figure 6As shown, the purified sample was concentrated, resulting in a higher sample concentration and improved sample purity.

[0046] Example 2 Recombinant collagen cell adhesion assay Cell source: HaCat (Servicebio, STCC11801P, human immortalized epidermal keratinocytes). Culture and passage methods were performed according to the cell manufacturer's instructions. Experimental group samples: Stable recombinant type XVII collagen (17A3 and 17A6 lyophilized sponges), prepared to a concentration of 10 mg / mL with sterile water, filtered through a 0.22 μm sterile filter for sterilization, and diluted to the required concentration in sterile DMEM. Positive control group: Fibronectin (FN, purchased from Solarbio, F8181). Blank control group: BSA. Fibronectin and BSA were dissolved in ultrapure water to a concentration of 5 mg / mL, filtered through a 0.22 μm sterile filter for sterilization, and diluted to 0.5 mg / mL in DMEM medium for use.

[0047] Coating Preparation: Add 100 μL of sample (sample, positive control, and blank control) to each well of a 96-well plate. Coat four wells with each sample and incubate at 37°C in a 5% CO2 incubator for 2 h. Remove excess coating solution from the wells, add 100 μL of 1% BSA-PBS solution, and incubate at 37°C in a 5% CO2 incubator for 2 h. After removing the liquid from the wells, wash three times with PBS, discard the washing solution, seal with sealing film, and store at 4°C for later use. Culture HaCat cells in a 37°C, 5% CO2 cell culture incubator. When the cells reach approximately 90% confluence, seed them. Use complete culture medium premixed with Hoechst-33342 fluorescent staining agent (10%) at a ratio of 1×10⁻⁶. 4 Seed cells into each well. After addition, cover with aluminum foil and incubate at 37°C and 5% CO2 for 1 hour. Measure three replicates; the fourth well is used to adjust microscope parameters and its measurement is not used.

[0048] Detection: At least 4×4 digital tiled images (fluorescence) were captured for each of the three wells using an inverted microscope. Each well was filled with PBS to form an "inverted meniscus," air bubbles were removed, and the plate was sealed with film. The plate was centrifuged at 300 g relative centrifugation (RCF) at 22°C (inverted plate) for 5 min. After centrifugation, the sealing film was discarded, and the supernatant was removed from the wells. The plate was washed once with D-PBS, followed by the addition of 100 μL of D-PBS. For each of the three wells, a total of 25 fluorescent tiled digital images were captured (at least a 4×4 matrix is ​​recommended, with 10% overlap). The cell count was calculated to be approximately 2400 to 3600 cells per sample (800–1200 cells / well × 3 wells). Three replicate samples were measured; the fourth well was used to adjust microscope parameters and its measurements were not used. The experiment showed that the coated samples exhibited stronger adhesion than the positive control group. The experimental results are as follows: Figure 7 As shown: a and b are 17A6, c and d are 17A3. The experimental results show that the experimental groups are significantly better than the blank control group. The adhesion-promoting effect on HaCat cells is similar to that of the positive control group, and there is a significant effect at the lowest sample concentration of 0.125 mg / ml.

[0049] Example 3 Recombinant collagen cell migration assay Cell sources were the same as in the cell adhesion experiment. Experimental group samples: stable recombinant type XVII collagen (17A3 and 17A6 lyophilized sponges), positive control group samples: human collagen (Sigma, catalog number C7774-5MG), blank experimental group: DMEM medium.

[0050] Experimental preparation: First, use a marker pen to draw evenly spaced horizontal lines on the back of a 6-well plate, approximately every 0.5cm to 1cm, passing through each well. Three lines should pass through each well. Add approximately 5×10⁻⁶ ppm of the filler material into each well. 5 Cells. Scratch assay: On the second day of cell culture, use a pipette tip, aligned with a ruler, to make a scratch as perpendicular as possible to the horizontal line on the back of the cell. The pipette tip must be vertical, not tilted. Wash the cells three times with PBS to remove the scratched cells. Add serum-free medium containing the test sample as the experimental group, with a concentration of 0.05%. Incubate at 37°C, 5% CO2. Take samples and photographs at 0h and 24h.

[0051] Data Processing: The scratch area of ​​each image was calculated using ImageJ image processing software. The cell migration rate for each group was calculated by dividing the total area of ​​migrating cells within the fixed scratch area by the initial area of ​​the fixed scratch area. A graph was plotted with time on the x-axis and the migration area ratio on the y-axis (in %), and the photos of the experimental and control groups at the initial 0-minute mark and at the end of the experiment were compared. One-way ANOVA was used to analyze the differences in data between the experimental groups, and the chi-square test was performed. The cell migration rate of the sample group was higher after 24 hours of experimentation. The experimental results are as follows: Figure 8 As shown: a and b are 17A6, c and d are 17A3. The experimental results show that the experimental groups are significantly better than the control group, and the effect is concentration-dependent, with the migration-promoting effect becoming more obvious as the concentration increases.

[0052] Example 4 Recombinant collagen cell activity assay Cells were derived from the same cell adhesion assay, and the Cell Counting Kit-8 (CCK-8) method was used to investigate the cytotoxicity of the stable recombinant type XVII collagen described in this invention. The stable recombinant type XVII collagen consisted of 17A3 and 17A6 (amino acid sequences as shown in SEQ ID No:6 and SEQ ID No:7).

[0053] The specific steps are as follows: (1) Material preparation: HaCat (immortalized human epidermal keratinocytes), the culture and passage methods are performed in accordance with the cell instructions.

[0054] Samples: Stable recombinant type XVII collagen 17A3 and 17A6 lyophilized sponges; after dissolving the samples in the specified solutions, they were sterilized by filtration through a 0.22 μm filter membrane and diluted with cell culture medium to 0.125 mg / ml, 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, and 10 mg / mL, with 3 replicate wells for each group.

[0055] Positive control group: Cell culture medium containing 10% DMSO; Blank control group: DMEM culture medium.

[0056] Experimental method: CCK8 cytotoxicity assay (2) Experimental procedure: Cell seeding: Collect cells in good growth condition and prepare a cell suspension using complete culture medium. Seed at 1.0 × 10⁶ cells per well. 4 / wells were inoculated into each group of wells in a 96-well plate and incubated in an incubator (37℃, 5% CO2) for 24 h.

[0057] Sample loading: Load samples when the cell deposition rate in the 96-well plate reaches 40%–60%. Solvent control group: Add 100 μL of cell culture medium to each well; Positive control group: Add 100 μL of culture medium containing 10% DMSO to each well; Sample group: Add 100 μL of culture medium containing the corresponding sample concentration to each well. After loading, incubate the 96-well plate in an incubator (37℃, 5% CO2) for 72 hours. After incubation, add 10 μL of CCK-8 solution to each well (avoid generating air bubbles). Incubate the plate in an incubator for 1–4 hours. Measure the absorbance at 450 nm using a microplate reader. (3) Data processing: Cell viability = [(As-Ab) / (Ac-Ab)] x 100% As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution, and drug solution); Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drugs); Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0058] Experimental results are as follows Figure 9 As shown: a represents the experimental results for 17A6, and b represents the experimental results for 17A3. The experimental results indicate that it is non-toxic to HaCat cells at all concentrations. Example 5 Stability test The stability of the obtained stable recombinant type XVII collagen 17A3 and 17A6 lyophilized sponges was investigated under repeated freeze-thaw cycles, different temperatures, and different pH conditions. The specific steps are as follows: (1) Repeated freeze-thaw cycles: The obtained stable recombinant type XVII collagen 17A3 and 17A6 lyophilized sponges were prepared into 1 mg / mL solutions with ddH2O, and incubated at -70℃ for 10 min, 99℃ for 10 min, and repeated three times. The samples were then analyzed by SDS-PAGE. The results are shown below. Figure 10 As shown in a.

[0059] As can be seen from the figure, the first sample on the left in each group of SDS-PAGE gel images is the sample that has not been repeatedly frozen and thawed. Among them, the stable recombinant type XVII collagen 17A3 and 17A6 have good stability under repeated freeze-thaw conditions and have not degraded.

[0060] (2) Stability at different pH levels: The obtained stable recombinant type XVII collagen 17A3 and 17A6 were prepared into 1 mg / mL solutions with ddH2O, and the pH was adjusted to different values ​​(4, 7, 9). The solutions were then incubated at 25°C for 7 days. Samples from days 1, 2, 3, 5, and 7 were subjected to SDS-PAGE. PAGE verification, experimental results are as follows: Figure 10 As shown in b, the three bands are pH4, pH7 and pH9 in sequence.

[0061] As can be seen from the figure, the stable recombinant type XVII collagen 17A3 and 17A6 are stable under pH conditions (4, 7 and 9), and the SDS-PAGE results show that there are no degradation bands.

[0062] (3) Stability at different temperatures: The obtained stable recombinant type XVII collagen 17A3 and 17A6 were prepared into 1 mg / mL solutions with ddH2O and placed at different temperatures (4°C, 25°C, 40°C, 60°C) for 7 days. Samples were taken at different times for SDS-PAGE. PAGE validation, validation results are as follows Figure 10 As shown in c, the band information for each day is 4°C, 25°C, 40°C, and 60°C, respectively.

[0063] As can be seen from the figure, the stable recombinant type XVII collagen 17A3 and 17A6 samples were sampled and analyzed by SDS-PAGE on days 1, 2, 3, 5 and 7. After 7 days, the protein content decreased slightly, but there was no overall degradation, indicating good stability.

[0064] In summary, the stable recombinant type XVII collagen 17A6 and 17A3 described in this invention have excellent stability.

[0065] In summary, this invention selects repetitive Gly-XY gene sequences from multiple triple-helix regions of human type XVII collagen for splicing or direct repetition to obtain stable recombinant type XVII collagen monomers. During selection, immune-dominant epitopes, unstable amino acid sequence triplets, easily hydrolyzed sites, and glycosylation sites are avoided, resulting in stable recombinant type XVII collagen with a molecular weight of 15-50 kDa. This stable recombinant type XVII collagen is beneficial for expression in Pichia pastoris and promotes adhesion and migration in human epithelial cells, enhancing cell activity and demonstrating excellent practicality. It maintains good stability under different temperatures and repeated freeze-thaw cycles. It shows great promise for future applications as a raw material in cosmetics and medical devices.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A stable recombinant type XVII collagen, characterized in that, The stable recombinant type XVII collagen comprises: a polypeptide with an amino acid sequence as shown in SEQ ID NO. 6; or, a polypeptide with an amino acid sequence as shown in SEQ ID NO.

7.

2. The gene encoding the stable recombinant type XVII collagen of claim 1, characterized in that, The nucleotide sequence of the gene is shown in any one of SEQ ID NO. 8-9.

3. An expression carrier, characterized in that, It includes the gene and empty vector as described in claim 2.

4. The expression vector according to claim 3, characterized in that, The empty vector is either the pPIC9K vector or the pPICZαA vector.

5. A genetically engineered bacterium, characterized in that, It includes the expression vector and empty host bacteria as described in claim 4.

6. The genetically engineered bacterium according to claim 5, characterized in that, The empty host bacteria is Pichia pastoris or Escherichia coli.

7. The genetically engineered bacterium according to claim 6, characterized in that, The genetically engineered bacteria are deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession numbers CGMCC No. 39095 and CGMCC No. 39094, respectively.

8. A method for preparing the genetically engineered bacteria according to claim 6, characterized in that, Includes the following steps: The expression vector described in claim 2 was linearized by enzyme digestion and transformed into Pichia pastoris. The Pichia pastoris genetically engineered strain expressing stable recombinant type XVII collagen was obtained by screening through the G418 resistance gradient.

9. The use of the recombinant human type XVII collagen of claim 1, the gene of claim 2, the expression vector of any one of claims 3-4, or the genetically engineered bacteria of any one of claims 5-7 in the preparation of pharmaceuticals, cosmetics, health products, or medical devices.

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