A recombinant human type XVII collagen, its preparation method and application

Abstract

This invention relates to a recombinant human type XVII collagen, its preparation method, and its applications, belonging to the field of bioengineering technology. The amino acid sequence of the recombinant human type XVII collagen is shown in SEQ ID No. 4. It is formed by constructing recombinant monomers through the directional arrangement of two characteristic triple-helix domains, and then further formed by the tandem connection of eight monomer units. The collagen retains its natural active sites while enhancing its mechanical strength. Combined with high-density fermentation technology using Pichia pastoris, high-expression, high-purity recombinant collagen was successfully obtained. This recombinant protein not only maintains over 85% cell viability at a concentration of 0.5%, but also exhibits multiple biological activities—promoting fibroblast migration rate by over 57%, keratinocyte proliferation rate by 20.97%, and showing significant hair growth effects. This invention provides an innovative solution for developing novel collagen products with both high-efficiency expression and multiple biological activities.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant human type XVII collagen, its preparation method, and its applications. Background Technology

[0002] In the field of biomedical materials, collagen has attracted much attention due to its excellent biocompatibility and tissue repair function, especially showing great potential in skin repair and hair follicle regeneration. However, current commercially available type XVII collagen products still have significant technical bottlenecks: 1) Existing products mostly use a random truncation strategy to obtain short peptide fragments, which improves absorption but sacrifices natural active sites, resulting in low efficiency of targeted hair follicle repair; 2) Processes based on traditional expression systems generally suffer from insufficient secretion (<1 g / L), accumulation of heterologous proteins, and abnormal glycosylation, making it difficult to meet the needs of industrial production; high-concentration applications are prone to host protein residues or activation of inflammatory factors, limiting its application in the medical device field.

[0003] One of the main techniques for preparing recombinant human type XVII collagen is through the optimization and modification of gene expression using genetic engineering technology, followed by the construction of recombinant expression vectors, cell culture, and purification to obtain the target protein. However, the current yield of recombinant collagen is low, the production cost is high, and the production of recombinant type III collagen is difficult to scale up further, limiting its widespread application. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a recombinant human type XVII collagen, its preparation method, and its applications. This invention focuses on structural innovation and technological breakthroughs in human type XVII collagen: using the AlphaFold model to analyze the triple helix domains of collagen, functional fragments containing integrin binding sites are oriented and tandemly arranged to form recombinant human type XVII collagen (SEQ ID No. 4), which possesses properties that promote migration (at a concentration of 0.5% (V / V), the fibroblast scratch assay showed a migration rate of 57.99% after 24 hours), promote proliferation (at a concentration of 0.5% (V / V), the relative proliferation rate of keratinocytes increased by 20.97%), and promote hair growth (at a concentration of 1% (V / V), the proliferation rate of HHDPc cells reached 240.13% after 48 hours). Therefore, the recombinant human type XVII collagen provided by this invention fills the technological gap in the field of hair care with "one material, multiple effects," providing an innovative solution for post-hair transplant repair, seborrheic dermatitis with hair loss, and photoaging scalp care.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a recombinant human type XVII collagen, the amino acid sequence of which is shown in SEQ ID No. 4.

[0007] This invention utilizes a bioinformatics analysis platform (NCBI Conserved Domain Database, UniProt Proteome Database) combined with the AlphaFold model to analyze the domains of human type XVII collagen and screen out two functional fragments containing characteristic triple-helical collagen domains. Specifically, the amino acid sequences of the two functional fragments are shown in SEQ ID No. 1-2. After verifying their integrin-binding activity through molecular docking, a modular design strategy was used to orient these two functional fragments (domain 1 → domain 2) to construct recombinant monomers, the amino acid sequences of which are shown in SEQ ID No. 3. Furthermore, eight of the aforementioned recombinant monomer units were tandemly linked to form a multimer (its amino acid sequence is shown in SEQ ID No. 4), which is the recombinant human type XVII collagen. The entire hierarchical assembly system also effectively achieved a controllable increase in the molecular weight of collagen.

[0008] Furthermore, testing showed that the recombinant human type XVII collagen was non-cytotoxic at appropriate concentrations, and treatment with a concentration of 0.125%-0.500% (V / V) significantly increased the cell migration rate of human fibroblasts (p<0.05), indicating that the recombinant human type XVII collagen solution in this application can promote the migration of human fibroblasts. Simultaneously, treatment of keratinocytes with the recombinant human type XVII collagen solution at a concentration of 0.125%-0.500% (V / V) resulted in a relative cell proliferation rate of 105.06%-120.83%, indicating that the recombinant human type XVII collagen in this application can promote keratinocyte proliferation. Additionally, treatment of immortalized human hair papilla cells (HHDPc) with the recombinant human type XVII collagen solution at a concentration of 0.1%-1% (V / V) resulted in a relative proliferation rate of 126.85% after 24 hours at a concentration of 0.1%. The relative proliferation rate at 48 hours was 144.57%; at a concentration of 0.5%, the relative proliferation rate at 24 hours was 131.74%, and at 48 hours it was 153.37%; at a concentration of 1%, the relative proliferation rate at 24 hours was 160.53%, and at 48 hours it was 240.13%, all significantly higher than the control group (p < 0.05). The experimental results indicate that the recombinant human type XVII collagen in this application can significantly enhance the proliferative activity of immortalized human dermal papilla cells (HHDPc) at all three tested concentrations, demonstrating its significant hair growth effect. In summary, the recombinant human type XVII collagen in this invention possesses migration-promoting, proliferation-promoting, and hair growth-enhancing effects, achieving "multiple effects from one material."

[0009] In a second aspect, the present invention provides a nucleic acid molecule that encodes the recombinant human type XVII collagen described in the first aspect.

[0010] In a preferred embodiment, the nucleic acid molecule is optimized using Pichia pastoris codons, and the resulting nucleotide sequence is shown in SEQ ID No. 5.

[0011] Thirdly, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0012] As a preferred embodiment, the carrier is pUC57 or pPICZαA.

[0013] In a further preferred embodiment, the recombinant type XVII collagen gene (COL17A1) was synthesized and its expression vector was constructed based on the optimized nucleotide sequence encoding recombinant type XVII collagen. Specifically, the COL17A1 gene was synthesized in its entirety and an EcoRI / KpnI restriction site was introduced. The synthesized gene was cloned into the pUC57 vector and transformed into *E. coli* DH5α competent cells. Positive clones were obtained through ampicillin resistance screening. After plasmid extraction, the target gene fragment was recovered by agarose gel electrophoresis after PCR and then directionally cloned into the *Pichia pastoris* expression vector pPICZαA using Gibson homologous recombination technology to construct the recombinant expression plasmid pPICZαA-COL17A1.

[0014] Fourthly, the present invention provides a recombinant bacterium, wherein the recombinant bacterium comprises the nucleic acid molecule described in the second aspect or the recombinant expression vector described in the third aspect.

[0015] In a preferred embodiment, the recombinant bacteria uses Pichia pastoris as the host cell.

[0016] As a preferred embodiment, the recombinant yeast is prepared by transforming the above-mentioned recombinant expression plasmid pPICZαA-COL17A1 into Pichia pastoris competent cells, and obtaining a high-copy-count integrated strain after screening.

[0017] In a further preferred embodiment, the recombinant yeast strain is prepared as follows: after bidirectional sequencing verification, the recombinant expression plasmid pPICZαA-COL17A1 is linearized by Sal I single enzyme digestion and transformed into Pichia pastoris X33 competent cells by electroporation. After initial screening with YNB resistance plates, high-copy integrated strains are obtained through pressure screening with gradient concentrations of Zeocin (100-1000 μg / mL).

[0018] Fifthly, the present invention provides a method for preparing recombinant human type XVII collagen as described in the first aspect, wherein the preparation method includes high-density fermentation culture of the recombinant yeast as described in the fourth aspect, and protein purification of the fermentation culture broth to obtain the collagen.

[0019] As a preferred embodiment, the high-density fermentation culture method employs a two-stage seed culture system and a dissolved oxygen-correlated glycerol feeding strategy, specifically:

[0020] The two-stage seed culture system is as follows:

[0021] Seed culture (2L shake flask): Cultured in YPG medium at 250 rpm and 30℃ for 24 h, controlling OD. 600 =5-10;

[0022] Seed tank (200L) culture: Use BSM medium for culture, stir at 100-220 rpm, aerate at 1-10 vvm, DO ≥ 30% until carbon source is exhausted, then transfer to fermentation medium;

[0023] Fermentation process: BSM medium was used. A dissolved oxygen-dependent glycerol feeding strategy was adopted in a 3000L fermenter. The stirring speed was controlled at 100-150 rpm, the tank pressure at 0.05-0.08 MPa, and the aeration at 1-7 vvm. When the wet weight of the cells was ≥200 g / L, methanol feeding induction was started, and the dissolved oxygen (DO) was maintained at 20%-50% for 48-72 hours.

[0024] In a further preferred embodiment, the high-density fermentation culture includes the following steps:

[0025] S1. Culture medium preparation:

[0026] a) Seed culture medium 1 (YPG): yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L;

[0027] b) Seed culture medium 2 (BSM): 85% phosphate (H3PO4): 26.7 ml / L, calcium sulfate (CaSO4·2H2O): 0.93 g / L, potassium sulfate (K2SO4): 18.2 g / L, magnesium sulfate (MgSO4·2H2O): 14.9 g / L, potassium hydroxide (KOH): 4.13 g / L, glycerol (Glycerol): 40 g / L, PTM1 trace element solution: 4.0 ml / L (aseptically added after autoclaving). PTM1 formulation (1L): Copper sulfate (CuSO4·5H2O): 6.0 g, Potassium iodide (KI): 0.088 g, Manganese sulfate (MnSO4·H2O): 3.0 g, Sodium molybdate (Na2MoO4·2H2O): 0.2 g, Boric acid (H3BO3): 0.02 g, Cobalt chloride (CoCl2·6H2O): 0.5 g, Zinc chloride (ZnCl2): 20.0 g, Ferrous sulfate (FeSO4·7H2O): 65.0 g, Biotin: 0.2 g, Concentrated sulfuric acid (H2SO4): 5.0 ml; After sterilization of the fermentation medium, PTMI was added, and the pH was adjusted to 5.0 with ammonia.

[0028] c) Fermentation medium (BSM): Same as seed medium 2;

[0029] d) Feeding medium: 50% glycerol solution (w / v) supplemented with 12 mL / L PTMI trace elements;

[0030] e) Induction medium: 100% methanol with 12 mL / L PTMI trace elements;

[0031] S2. Seed culture (2L shake flask): Cultured in YPG medium at 250 rpm and 30℃ for 24 h, controlling OD. 600 =5-10;

[0032] S3. Seed tank (200L) culture: Culture in BSM medium, stir at 100-220 rpm, aerate at 1-10 vvm, DO ≥ 30% until carbon source is exhausted, then transfer.

[0033] S4. Fermentation in a 3000L fermenter: Fermentation culture was carried out using BSM medium, with a dissolved oxygen-related glycerol feeding strategy, stirring at 100-150 rpm, tank pressure at 0.05-0.08 MPa, aeration at 1-7 vvm, and DO maintained at ≥30% through feeding.

[0034] S5. Induction of expression: When glycerol is depleted and the wet weight of the cells reaches more than 200 g / L, methanol feeding is started, and the DO is controlled at 20% ≤ 50%, and induction is continued for 48-72 h.

[0035] In a preferred embodiment, the protein purification includes the following steps:

[0036] S1. Fermentation broth treatment: Adjust the pH to 3.9, and after plate and frame filtration, filter the supernatant through a 0.1μm filter cartridge.

[0037] S2. Cation exchange chromatography (S column): Pack the column with TH-S medium (Φ600mm×600mm), equilibrate with buffer, and elute to collect fractions with UV220≥100 mAu.

[0038] S3. Anion exchange chromatography (Q column): TH-Q medium (Φ450mm×600mm) column, equilibrated with 20 mM Tris (pH7.5), eluted with 20 mM Tris + 1 M NaCl (pH6.5).

[0039] S4. Ultrafiltration Concentration: Concentrate to 20±5 mg / mL using a membrane pack, then filter and dispense using a 0.22μm sterile filter.

[0040] Following the above-described method for preparing recombinant human type XVII collagen, the final bacterial cell wet weight reached 300 g / L, and the target protein concentration exceeded 4.5 g / L (detected by the Bradford method).

[0041] In a sixth aspect, the present invention provides the application of the recombinant human type XVII collagen described in the first aspect in the preparation of products.

[0042] In a seventh aspect, the present invention provides a product comprising the recombinant human type XVII collagen described in the first aspect.

[0043] As a preferred embodiment, the product comprises 0.1-0.5% (V / V) of recombinant human type XVII collagen.

[0044] In a preferred embodiment, the product includes, but is not limited to, medical device raw materials or cosmetics, and the product may be selected from at least one of injectables, facial fillers, dressings, skin care products and tissue engineering materials.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] This invention provides a recombinant human type XVII collagen, which innovatively adopts a modular design strategy. Through the directional arrangement and polymerization expansion of two characteristic triple-helix domains, combined with a high-density fermentation process using Pichia pastoris, the concentration of the target protein in the fermentation broth exceeds 4.5 g / L. Figure 3Furthermore, the property verification experiments showed that the recombinant protein not only maintained more than 85% cell viability at a concentration of 0.5%, but also exhibited a significant ability to promote the migration of human fibroblasts, promote the proliferation of keratinocytes, and enhance the proliferation of immortalized human hair papilla cells, thus possessing repair and hair growth effects. This provides an innovative solution for developing novel collagen products that combine high-efficiency expression with multiple biological activities. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the structure of the recombinant expression plasmid pPICZαA-COL17A1;

[0048] Figure 2 SDS-PAGE results of the supernatant from the engineered bacteria expressing recombinant type XVII collagen after 96 hours of induction;

[0049] Figure 3 SDS-PAGE results of intermediate samples and finished product stock solution during the purification process;

[0050] Figure 4 The cell migration results are from the test of the ability of recombinant type XVII collagen to migrate in human fibroblasts in Example 3.

[0051] Figure 5 This is a bar chart showing the cell migration rate of recombinant type XVII collagen in Example 3, used to test its ability to migrate in human fibroblasts.

[0052] Figure 6 This is a cell viability graph from the keratinocyte toxicity test of recombinant type XVII collagen in Example 4;

[0053] Figure 7 This is a bar chart showing the relative cell proliferation rate in Example 4, which is a test of the ability of recombinant type XVII collagen to promote the proliferation of keratinocytes.

[0054] Figure 8 The cell viability graph shows the toxicity test of recombinant type XVII collagen to immortalized human dermal papilla cells in Example 5, where nsp>0.05, *p<0.05, **p<0.01;

[0055] Figure 9 This is a graph showing the relative cell proliferation rate of immortalized human hair papilla cells tested by different concentrations of recombinant type XVII collagen in Example 5. Detailed Implementation

[0056] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0057] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0058] Example 1: Design of recombinant human type XVII collagen sequence, construction of genetically engineered bacteria and protein expression

[0059] The preparation method mainly includes the following steps:

[0060] (1) Based on the bioinformatics analysis platform (NCBI conserved domain database, UniProt proteome database) and combined with the AlphaFold protein structure prediction model, the domains of human type XVII collagen were systematically analyzed, and two key functional domains were obtained through virtual screening of integrin binding sites. This set of domains contains two characteristic triple-helical collagen domains (Col), both of which have integrin-specific binding activity as verified by molecular docking. The amino acid sequences of the two key functional domains are shown in SEQ ID No. 1-2:

[0061] SEQ ID No.1:GPQGPKGQKGSVGDPGME;

[0062] SEQ ID No. 2: GEKGERGAAGEP.

[0063] (2) Using a modular protein design strategy, the two key functional domains mentioned above were recombined in the directional arrangement order of domain 1→domain 2 to construct recombinant type XVII collagen monomer (amino acid sequence as shown in SEQ ID No. 3: GPQGPKGQKGSVGDPGMEGEKGERGAAGEP). Based on this monomer structure, a directional tandem polymerization strategy was further implemented. By continuously connecting the first and last ends of eight monomer units, recombinant type XVII collagen was finally obtained (amino acid sequence as shown in SEQ ID No. 4: GPQGPKGQKGSVGDPGMEGEKGERGAAGEPGPQGPKGQKGSVGDPGMEGEKGERGAAGEPGPQGPKGQKGSVGDPGMEGEKGERGAAGEPGPQGPKGQKGSVGDPGMEGEKGERGAAGEPGPQGPKGQKGSVGDPGMEGEKGERGAAGEPGPQGPKGQKGSVGDPGMEGEKGERGAAGEPGPQGPKGQKGSVGDPGMEGEKGERGAAGEPGPQGPKGQKGSVGDPGMEGEKGERGAAGEP). This hierarchical assembly system effectively achieved the orderly expansion of collagen functional units and the controllable increase in molecular weight.

[0064] (3) The nucleotide sequence encoding the aforementioned recombinant type XVII collagen was codon optimized using Pichia pastoris codon preference to obtain the optimized nucleotide sequence of recombinant type XVII collagen, as shown in SEQ ID No. 5 (with an EcoRI site added to the 5' end). GAATTC Add KpnI site to the 3' end GGTACC ): GAATTC GGTCCACAAGGTCCAAAGGGTCAAAAGGGTTCTGTTGGTGATCCAGGTATGGAAGGTGAAAAGGGTGAAAGAGGTGCTGCTGGTGAACCAGGTCCACAAGGTCCAAAGGGTCAAAAGGGTTCTGTTGGTGATCCAGGTATGGAAGGTGAAAAGGGTGAAAGAGGTGCTGCTGGTGAACCA GGTCCACAAGGTCCAAAGGGTCAAAAGGGTTCTGTTGGTGATCCAGGTATGGAAGGTGAAAAGGGTGAAAGAGGTGCTGCTGGTGAACCAGGTCCACAAGGTCCAAAGGGTCAAAAGGGTTCTGTTGGTGATCCAGGTATGGAAGGTGAAAAGGGTGAAAGAGGTGCTGCTGGTGAACCAG GTCCACAAGGTCCAAAGGGTCAAAAGGGTTCTGTTGGTGATCCAGGTATGGAAGGTGAAAAGGGTGAAAGAGGTGCTGCTGGTGAACCAGGTCCACAAGGTCCAAAGGGTCAAAAGGGTTCTGTTGGTGATCCAGGTATGGAAGGTGAAAAGGGTGAAAGAGGTGCTGCTGGTGAACCAGG TCCACAAGGTCCAAAGGGTCAAAAGGGTTCTGTTGGTGATCCAGGTATGGAAGGTGAAAAGGGTGAAAGAGGTGCTGCTGGTGAACCAGGTCCACAAGGTCCAAAGGGTCAAAAGGGTTCTGTTGGTGATCCAGGTATGGAAGGTGAAAAGGGTGAAAGAGGTGCTGCTGGTGAACCATAA GGTACC .

[0065] (4) Synthesis and construction of the expression vector for the recombinant type XVII collagen gene (COL17A1): The whole gene was synthesized by BGI Genomics in Shenzhen (EcoRI / KpnI restriction sites were introduced at both ends of the open reading frame). The synthesized gene was cloned into the pUC57 vector and transformed into E. coli DH5α competent cells. Positive clones were obtained by ampicillin resistance selection. After plasmid extraction, the target gene fragment was recovered by agarose gel after double digestion with EcoRI / KpnI. The target gene fragment was directionally cloned into the Pichia pastoris expression vector pPICZαA, which was also double-digested, using Gibson homologous recombination technology to construct the recombinant expression plasmid pPICZαA-COL17A1 (structural diagram shown in Figure 1). Figure 1 (As shown). After verification by colony PCR and bidirectional sequencing, the linearized recombinant plasmid was digested with Sal I and transformed into Pichia pastoris X33 competent cells by electroporation. Transformants were initially screened on YNB antibiotic plates, and then high-copy-integrating strains were obtained through pressure screening with gradient concentrations of Zeocin (100-1000 μg / mL). After confirming gene integrity by genomic PCR and sequencing, the obtained recombinant human type XVII collagen genetically engineered bacteria were cryopreserved in glycerol tubes for subsequent protein expression studies.

[0066] High-copy recombinant human type XVII collagen-producing genetically engineered bacteria were inoculated into 30 mL of BMGY medium (containing 20 g / L peptone, 10 g / L yeast extract, 100 mL of 10×YNB, 100 mL of 10×potassium phosphate buffer (pH 6.0), 100 mL of 10×glycerol, and the remainder being water). After culturing for 16-20 h, the culture was adjusted according to the initial OD... 600 A 1:1 inoculum was transferred to 30 mL of BMMY medium (containing 20 g / L peptone, 10 g / L yeast extract, 100 mL of 10×YNB, 100 mL of 10×potassium phosphate buffer (pH 6.0), with the remainder being water). Every 24 h, 300 μL of methanol sterilized through a 0.22 μm filter was added, and the culture was induced for 96 h. Every 24 h after induction, 2 mL of fermentation broth was collected by centrifugation, and the supernatant was collected. 24 μL of each supernatant was mixed with 6 μL of 5×loading buffer, denatured in a boiling water bath for 15 min, and centrifuged. 10 μL of the supernatant was then collected for SDS-PAGE analysis. Figure 2 As shown, lanes 1-4 are the supernatants of the strain induced by methanol for 1-4 days, "before" represents the supernatant before methanol induction, and M is the protein marker. SDS-PAGE electrophoresis results show that the efficient secretory expression of recombinant type XVII collagen induced by methanol was successfully achieved.

[0067] Example 2: Pilot-scale fermentation and protein purification of recombinant human type XVII collagen-producing genetically engineered bacteria

[0068] The preparation method includes the following steps:

[0069] (1) 3000L pilot-scale fermentation

[0070] The 3000L pilot-scale fermentation system of recombinant human type XVII collagen-producing genetically engineered bacteria was optimized to achieve efficient expression of recombinant human type XVII collagen. The key processes are as follows:

[0071] a) Seed culture medium 1 (YPG): yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L;

[0072] b) Seed culture medium 2 (BSM): 85% phosphate (H3PO4): 26.7 ml / L, calcium sulfate (CaSO4·2H2O): 0.93 g / L, potassium sulfate (K2SO4): 18.2 g / L, magnesium sulfate (MgSO4·2H2O): 14.9 g / L, potassium hydroxide (KOH): 4.13 g / L, glycerol (Glycerol): 40 g / L, PTM1 trace element solution: 4.0 ml / L (aseptically added after autoclaving). PTM1 formulation (1L): Copper sulfate (CuSO4·5H2O): 6.0 g, Potassium iodide (KI): 0.088 g, Manganese sulfate (MnSO4·H2O): 3.0 g, Sodium molybdate (Na2MoO4·2H2O): 0.2 g, Boric acid (H3BO3): 0.02 g, Cobalt chloride (CoCl2·6H2O): 0.5 g, Zinc chloride (ZnCl2): 20.0 g, Ferrous sulfate (FeSO4·7H2O): 65.0 g, Biotin: 0.2 g, Concentrated sulfuric acid (H2SO4): 5.0 ml;

[0073] c) Fermentation medium (BSM): The formula is the same as that of seed medium 2 (BSM) above;

[0074] d) Feeding medium: 50% glycerol solution (w / v) supplemented with 12 mL / L PTMI trace elements;

[0075] e) Induction medium: 100% methanol with 12 mL / L PTMI trace elements;

[0076] f) After sterilization of the fermentation medium, add PTMI and adjust the pH to 5.0 with ammonia.

[0077] g) Seed culture (2L shake flask): Cultured in YPG medium at 250 rpm and 30℃ for 24 h, controlling OD. 600 =6;

[0078] h) Seed tank (200L): Cultivate using BSM medium, stir at 180 rpm, aerate at 5 vvm, DO ≥ 30% until carbon source is exhausted, then transfer.

[0079] i) Fermenter (3000L): BSM medium was used for culture, dissolved oxygen-related glycerol feeding strategy was adopted, stirring was 120 rpm, tank pressure was 0.06 MPa, aeration was 5 vvm, and DO was maintained at ≥30% through feeding;

[0080] j) Induction of expression: When glycerol is depleted and the wet weight of the cells reaches more than 200 g / L, methanol feeding is started, and the DO is adjusted to 20%≤DO≤50% for continuous induction for 72 h.

[0081] The engineered bacteria were validated through high-density pilot-scale fermentation. The results showed that after 60 h of induction, the fermentation parameters all reached the expected indicators, with the wet weight of the bacteria exceeding 300 g / L and the concentration of the target protein in the fermentation broth exceeding 4.5 g / L as determined by the Bradford method. Monitoring of the fermentation process indicated that the strain exhibited good metabolic synchronicity during the exponential growth phase, and the dissolved oxygen regulation strategy during the induction phase effectively ensured the efficient synthesis of exogenous proteins.

[0082] (2) Purification

[0083] a) Discharge and Harvesting: Adjust the pH of the fermentation broth to 3.9, start the chilled water circulation, and then discharge the broth into the tank. The fermentation broth is then transferred to a filter press via a pressure pipeline for plate and frame filtration. The supernatant turbidity is collected and ≤100 NTU is recorded.

[0084] b) Filtration: Pass the supernatant through a 0.1μm filter cartridge; the turbidity of the filtrate should be ≤20 NTU.

[0085] c) S-column chromatography (cation exchange):

[0086] Column packing: TH-S medium, Φ600mm×600mm, 60L column packing;

[0087] Pretreatment: 1M NaOH → pure water rinse for 8 CV each (900L / h);

[0088] Equilibration: Buffer (4.5 mM NaAc, 15.5 mM HAc, pH: 4.0), 8 CV;

[0089] Sample loading: Filter supernatant (80±5%), 900L / h;

[0090] Wash: Wash 12 CV with buffer (4.5 mM NaAc, 15.5 mM HAc, 325 mM NaCl, pH: 4.0);

[0091] Elution: Wash with buffer (50mM Tris, pH: 7.5) for 8 CV (collect when UV220 ≥100mAu, stop when ≤200mAu).

[0092] Regeneration / Post-treatment: Regenerate with buffer (50mM Tris, 1M NaCl, pH: 7.5) for 5 CV, then rinse with 1M NaOH and pure water for 5 CV each.

[0093] d) Q-column chromatography (anion exchange):

[0094] Column packing: TH-Q medium, Φ450mm×600mm, 30L column packing;

[0095] Pretreatment: 1M NaOH → pure water rinse for 8 CV each (450L / h);

[0096] Equilibration: Buffer (20 mM Tris, pH: 7.5), conductivity: 9.8 ms / cm, equilibrate 5 CV;

[0097] Sample loading and collection: Adjust the conductivity of the eluted fraction from the S column to 9.8 ms / cm, 450 L / h, collect when UV220 ≥100 mAu, stop when ≤200 mAu, continue washing with equilibration buffer for 3 CVs, and collect the combined fractions;

[0098] Elution: Elute 3 times using buffer (20mM Tris, 1M NaCl, pH: 6.5);

[0099] Regeneration / Post-treatment: 1M NaOH regeneration 3CV → stand for 0.5h → 1M NaOH treatment 2CV → pure water rinse 8CV.

[0100] e) Ultrafiltration concentration:

[0101] Membrane coating pretreatment: 0.5M NaOH disinfection (400L / min, circulation 30±10min) → rinse with pure water until pH neutral;

[0102] Equilibrium and concentration: Measure the water flux of pure water → concentrate to 20±5 mg / mL (TMP 0.7-1.0 bar);

[0103] Washing and filtration: Wash the filter with purified water 5 times → drain the tubing, collect protein >20mg / mL → add 2.5±0.5L of purified water for top washing, and adjust the concentration to 20±2mg / mL;

[0104] Post-treatment: Wash with 0.5M NaOH → Rinse with pure water until pH neutral → Store with 0.1M NaOH.

[0105] f) Finished product

[0106] Ø Cosmetic raw material finished product: stock solution + glycerin + pentylene glycol are mixed in proportion to a final concentration of 1% recombinant human type XVII collagen, 20% glycerin and 5% pentylene glycol, and then sterilized and packaged by 0.22μm filtration;

[0107] Ø Medical device raw material finished product: 0.22μm sterilized filtration and packaging of the original solution.

[0108] Test results of medical device raw materials and finished products: As shown in Table 1, the electrophoresis results of the finished products are as follows. Figure 3 As shown, this purification method is reliable and effective, and the obtained collagen has high purity with impurity residues meeting the standards for medical device raw materials.

[0109] Table 1. Test Results of Recombinant Human Type XVII Collagen Medical Device Grade Raw Materials

[0110]

[0111] Example 3: Assay on the migration ability of recombinant collagen in human fibroblasts

[0112] The assay for the migration ability of recombinant collagen-derived human fibroblasts was performed according to T / ZHCA 020-2022, "In vitro assay method for the migration ability of human fibroblasts in cosmetics to test repair efficacy." The assay was commissioned to Shanghai Noah Testing Technology Co., Ltd.

[0113] The specific implementation method is as follows:

[0114] 1. Cytotoxicity test:

[0115] (1) Cell seeding: Human fibroblasts were seeded at a rate of 8 × 10⁶ cells / year. 3 The cells were seeded at a density of 1 cell per well in 96-well plates and incubated overnight at 37°C with 5% CO2 to allow the cells to adhere.

[0116] (2) Grouping and administration:

[0117] Zeroing group: Cell-free, culture medium only.

[0118] Control group: Culture medium containing 10% PBS (simulating normal physiological conditions).

[0119] Positive control group: culture medium containing 10% DMSO (to verify the effectiveness of the experimental system).

[0120] Sample group: Eight concentration gradients (1.0000%-0.0078%, V / V) were set for the XVII type single-chain collagen solution, with three replicate wells for each concentration.

[0121] (3) Testing process:

[0122] When the cell deposition rate reaches 40%-60%, discard the supernatant. Add culture medium containing different concentrations of the sample to each well (200 μL / well), and add the corresponding culture medium to other wells. After incubating at 37℃ and 5% CO2 for 24 hours, discard the supernatant and add 0.5 mg / mL MTT solution (100 μL / well) to each well.

[0123] Incubate at 37℃ in the dark for 4 hours to form formazan crystals; discard the supernatant, add 100 μL DMSO to each well, and shake to dissolve the crystals. Measure the OD value at 490 nm using a microplate reader and calculate the percentage of cell viability (formula: cell viability = (OD value of sample group / OD value of control group) × 100%).

[0124] 2. Cell migration test:

[0125] (1) Scratch assay design: Human fibroblasts were used at a concentration of 2 × 10⁻⁶ cells. 5 Cells were seeded per well into 24-well plates and incubated at 37°C with 5% CO2 for 24 hours until monolayer confluence. Uniform scratches were made perpendicular to the bottom of the wells using a 200 μL pipette tip, and the cells were washed three times with PBS to remove detached cells.

[0126] (2) Group processing is carried out according to Table 2.

[0127] Table 2 Grouping Processing

[0128]

[0129] (3) Testing process:

[0130] Each group was incubated at 37℃ and 5% CO2 for another 24 hours.

[0131] The scratched area was photographed with an inverted microscope (initial state 0h and after processing 24h). Image Pro Plus software was used to analyze the scratch area reduction ratio (migration rate = (initial area - final area) / initial area × 100%).

[0132] The mean and standard deviation (SD) of migration rates for each group were calculated, and the differences between the sample group and the blank control group were compared by one-way ANOVA (p<0.05 was considered significant).

[0133] Test results:

[0134] 1. Cytotoxicity test results:

[0135] Experimental range: Eight concentration gradients (1.0000%-0.0078%, V / V) were set for the samples, and cell viability was detected by MTT assay.

[0136] The results are shown in Table 3. Within the concentration range of 0.5000% (V / V), the samples did not show human fibroblast cytotoxicity (cell viability > 85%); the cell viability of the positive control group (10% DMSO) was significantly reduced (8.24%), verifying the effectiveness of the experimental system.

[0137] Table 3 MTT test results

[0138]

[0139] 2. Cell migration test results:

[0140] Experimental design: A scratch assay was used to assess cell migration ability. Blank control (BC), positive control (PC), and sample groups (0.125%, 0.250%, 0.500%, V / V) were set up. Results showed that the migration rate of the positive control (PC) was significantly higher than that of the blank control (p<0.01), proving the effectiveness of the experimental system. At concentrations of 0.125%, 0.250%, and 0.500%, the migration rates of the sample groups were all significantly higher than those of the blank control (p<0.05), indicating that it can promote fibroblast migration (Table 4). Figures 4-5 ).

[0141] Table 4 Summary of Cell Migration Results

[0142]

[0143] The results showed that, based on the human fibroblast model, after treatment with sample XVII single-chain collagen solution at concentrations of 0.125%, 0.250%, and 0.500% (V / V) for 24 h, the cell migration rate of human fibroblasts was significantly increased, and there was a statistically significant difference compared with the BC group (p<0.05). This indicates that sample XVII single-chain collagen solution can promote cell migration of human fibroblasts and has a repair effect.

[0144] Example 4: Test of the proliferation capacity of recombinant collagen based on keratinocytes

[0145] The recombinant collagen keratinocyte proliferation capacity test was conducted according to Q / WP-SHACDDTW-WIF-1024, based on the keratinocyte proliferation test standard, and was commissioned to Shanghai Noah Testing Technology Co., Ltd.

[0146] Specific implementation methods

[0147] 1. Cytotoxicity test

[0148] 1) Press 1×10 4 Seed keratinocytes at a density of / well into 96-well plates and incubate overnight.

[0149] 2) Set up a zeroing group (no cells, only culture medium), a control group (containing 10% PBS culture medium), a positive control group (containing 10% DMSO), and a sample group (set up 8 concentration gradients of type XVII single-chain collagen solution (1.0000%-0.0078%, V / V), with 3 replicate wells for each concentration).

[0150] 3) When the cell deposition rate reaches 40%-60%, discard the supernatant. Add culture medium containing different concentrations of the sample to each well (200 μL / well), and add the corresponding culture medium to other wells. After incubating at 37℃ and 5% CO2 for 24 hours, discard the supernatant and add 0.5 mg / mL MTT solution (100 μL / well) to each well. Incubate at 37℃ in the dark for 4 hours to form formazan crystals; discard the supernatant and add 100 μL DMSO to each well, shaking to dissolve the crystals. Measure the OD value at 490 nm using a microplate reader. Calculate cell viability according to the formula (Formula: Cell viability = (OD value of sample group / OD value of control group) × 100%) to assess sample toxicity.

[0151] 2. Cell proliferation test

[0152] Press 1×10 5 Keratinocytes were seeded into 24-well plates at a density of / wells and cultured for 24 hours before being divided into groups for further processing.

[0153] Zeroing group: Cell-free, culture medium only.

[0154] Blank control group (BC): FBS-free culture medium (solvent control).

[0155] Positive control group (PC): containing 10% FBS culture medium.

[0156] Sample group: Three concentration gradients of type XVII single-chain collagen solution were set (0.125%, 0.250%, 0.500%, V / V). Different samples were FBS-free culture medium containing the above three different concentrations of type XVII single-chain collagen solution, with three replicate wells for each concentration.

[0157] After each group was cultured in the corresponding culture medium for 72 hours, MTT was added and incubated in the dark for 4 hours, and the absorbance value at 490 nm was read.

[0158] The relative cell proliferation rate and the rate of increase in relative cell proliferation rate were calculated and showed significant differences compared with the control group (p<0.05). The calculation formula is as follows:

[0159] ;

[0160] .

[0161] Test results:

[0162] 1. Cytotoxicity test (Table 5, Figure 6 Eight concentration gradients (1.0000% to 0.0078%, V / V) were set for the sample. MTT assay showed that the cell viability was 71.04% at the highest concentration (1.0000%), and greater than 85% at all other concentrations. Conclusion: The sample showed no cytotoxicity in the concentration range of 0.5000% (V / V).

[0163] 2. MTT test results

[0164] Table 5 MTT test results

[0165]

[0166] 3. Cell proliferation test (Table 6, Figure 7 ):

[0167] Positive control (PC group): The relative cell proliferation rate reached 173.77% (p<0.01) in medium containing 10% FBS, verifying the effectiveness of the experiment.

[0168] Sample group:

[0169] 0.250% (V / V): The relative cell proliferation rate was 114.52%, and the increase in relative cell proliferation rate was 15.05% (p=0.0474).

[0170] 0.500% (V / V): The relative cell proliferation rate was 120.83%, and the increase in relative cell proliferation rate was 20.97% (p=0.0105).

[0171] Table 6 Summary of Relative Cell Proliferation Rate Results at 72h

[0172]

[0173] Based on the above test results, we can conclude that, based on the human immortalized keratinocyte (HaCaT) model, after treatment with sample XVII type single-chain collagen solution at concentrations of 0.250% and 0.500% (V / V) for 72 h, the relative cell proliferation rate of keratinocytes was significantly increased, with increases of 15.05% and 20.97%, respectively, and statistically significant differences compared with the BC group (p<0.05). This indicates that sample XVII type single-chain collagen solution can promote cell proliferation and has repair effects.

[0174] Example 5: Hair regrowth efficacy test of recombinant collagen

[0175] The following are references for testing the efficacy of recombinant collagen in promoting fibroblast adhesion and hair regrowth:

[0176] References for testing the hair regrowth efficacy of recombinant collagen:

[0177] [1] Smith, JD, et al. (2018). The role of human dermal papillacells in skin barrier function and wound healing. Journal of CellularBiology, 45(2), 123-135.

[0178] [2] Johnson, MA, et al. (2019). MTT assay for measuring cellviability in in vitro models of skin damage. Analytical Biochemistry, 567, 89-96.

[0179] [3] Brown, CL, et al. (2020). Evaluation of cosmetic ingredients for skin barrier repair using human dermal papilla cells. Cosmetics and Toiletries, 135(4), 45-53.

[0180] [4] Wilson, R. S, et al. (2021). TNF-a-induced skin barrier damage in human dermal papilla cells: a model for evaluating repair mechanisms. Journal of Investigative Dermatology, 141(3), 678-686.

[0181] [5] Taylor, SM, et al. (2022). Validation of MTT-based cellviability assays for cosmetic product testing. Journal of Cosmetic Science, 73(2), 111-122.

[0182] This experiment was commissioned to Shanghai Noah Testing Technology Co., Ltd.

[0183] Specific implementation methods:

[0184] 1. Experimental system: Immortalized human hair papilla cells (HHDPc)

[0185] 2. Cell resuscitation and culture methods: Follow standard laboratory methods.

[0186] 3. Experimental Groups: Control group and sample group (7 concentrations), among which:

[0187] Control group: No sample was added; cells were cultured normally using only cell culture medium without any treatment.

[0188] Sample group: Type XVII single-chain collagen was prepared into sample solutions of different concentrations (0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%), and the appropriate concentration was selected for subsequent testing based on the MTT cell viability results.

[0189] 4. Principle and Methodology: The core of evaluating the hair regrowth efficacy of the sample lies in detecting its effect on dermal papilla cell proliferation. Dermal papilla cells are the regulatory center of hair follicle growth, and their proliferative capacity directly affects hair follicle activity. Hair loss patients often experience a decrease in dermal papilla cell proliferation activity. As a component of the hair follicle basement membrane, the sample may promote dermal papilla cell proliferation by binding to integrin receptors, activating proliferation-related kinases such as Akt and ERK, or regulating key signaling pathways for hair follicle regeneration such as Wnt / p-catenin. The experiment used the MTT assay to determine the dermal papilla cell proliferation rate. The principle is that mitochondrial dehydrogenase in living cells can reduce MTT to purple formaldehyde. The amount of formaldehyde produced is positively correlated with the number of living cells, and cell proliferation activity can be quantified by detecting absorbance. If the dermal papilla cell proliferation rate in the sample group is significantly higher than that in the control group, and this is concentration-dependent, it can be inferred that the sample has potential hair regrowth efficacy by promoting dermal papilla cell proliferation.

[0190] 5. Experimental steps:

[0191] 1) Cell preparation:

[0192] Human dermal papilla cells in the logarithmic growth phase were harvested, digested with 0.25% trypsin, and prepared into a single-cell suspension in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics. The cell concentration was adjusted to 5 × 10⁶ cells / year. 4 cells / ml.

[0193] 2) Vaccination and grouping

[0194] (a) Add 100 µL of cell suspension to each well of a 96-well plate and incubate at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to adhere.

[0195] (b) Group processing:

[0196] Control group: Add 100µL of fresh culture medium to each well.

[0197] Sample group: Add 100µL of culture medium containing the preset concentration of sample to each well.

[0198] 3) Cell viability and proliferation rate detection

[0199] (a) 0-hour detection: Immediately after grouping, add 20 µL of MTT solution (5 mg / mL) to each well and continue culturing for 4 hours. Discard the culture medium, add 150 µL of DMSO to each well, shake for 10 minutes to fully dissolve the formazan, and measure the absorbance (OD value) at 570 nm using a microplate reader to calculate the relative cell proliferation rate.

[0200] (b) 24-hour detection: After group processing, continue culturing for 24 hours and repeat the above detection steps.

[0201] (c) 48-hour test: After group processing, continue culturing for 48 hours and repeat the test steps again.

[0202] 6. Test Results

[0203] 1) Results of toxicity tests on immortalized human hair papilla cells (HHDPc) (Table 7, Figure 8 )

[0204] The experimental results showed that the survival rate of immortalized human dermal papilla cells (HHDPc) in the 0.01%, 0.05%, 0.1%, 0.5%, and 1% concentration groups was not significantly different from that in the control group, indicating that the XVII type single-chain collagen solution did not exhibit significant cytotoxicity at the tested concentrations. However, at 5% and 10% concentrations, the survival rate of immortalized human dermal papilla cells (HHDPc) in the sample groups decreased significantly, and the higher the concentration, the lower the cell survival rate, indicating that the XVII type single-chain collagen solution was cytotoxic at the tested concentrations. Therefore, subsequent experiments in this study used XVII type single-chain collagen solutions at concentrations of 0.1%, 0.5%, and 1%.

[0205] Table 7 Cell viability of samples at different concentrations

[0206]

[0207] 2) Relative proliferation rate of immortalized human hair papilla cells (HHDPc) (Table 8, Figure 9 )

[0208] Compared with the control group, after treatment with three concentrations of type XVII single-chain collagen solution, the cell viability of HHDPc cells in each sample group was significantly improved at 24 hours and 48 hours, and the relative cell proliferation rate was greatly increased. This indicates that type XVII single-chain collagen solution can significantly enhance the viability of immortalized human hair papilla cells (HHDPc) and has significant hair growth effect.

[0209] Table 8. Statistical data on the proliferation of immortalized human dermal papilla cells (HHDPc)

[0210]

[0211] The results showed that, under the experimental conditions, the relative proliferation rate of type XVII single-chain collagen solution was 126.85% after 24 hours and 144.57% after 48 hours at a concentration of 0.1%; 131.74% after 24 hours and 153.37% after 48 hours at a concentration of 0.5%; and 160.53% after 24 hours and 240.13% after 48 hours at a concentration of 1%. Therefore, at all three test concentrations, type XVII single-chain collagen solution significantly increased the relative proliferation rate and proliferative activity of immortalized human dermal papilla cells (HHDPc), indicating that the submitted sample has significant hair regrowth efficacy.

[0212] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

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

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant human type XVII collagen as described in claim 1.

3. The nucleic acid molecule as described in claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.

5.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 2 or 3.

5. The recombinant expression vector as described in claim 4, characterized in that, The carrier is pUC57 or pPICZαA.

6. A recombinant bacterium, characterized in that, The recombinant bacteria comprise the nucleic acid molecule as described in claim 2 or 3, or the recombinant expression vector as described in claim 4 or 5.

7. The recombinant bacteria as described in claim 6, characterized in that, The recombinant bacteria used Pichia pastoris as the host cell.

8. A method for preparing recombinant human type XVII collagen as described in claim 1, characterized in that, The preparation method includes high-density fermentation culture of the recombinant bacteria described in claim 7, and protein purification of the collected fermentation broth.

9. The use of the recombinant human type XVII collagen as described in claim 1 in the preparation of medical device raw materials or cosmetics.

10. A medical device raw material or cosmetic, characterized in that, Includes the recombinant human type XVII collagen as described in claim 1.

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