Recombinant human XVII type collagen as well as preparation method and application thereof

Abstract

The invention belongs to the field of bioengineering, and discloses a recombinant XVII type collagen as well as a preparation method and application thereof. A natural long fragment from a collagen structural domain of the XVII type collagen C15 is expressed through escherichia coli, and the recombinant XVII type collagen with high soluble expression quantity is obtained. The recombinant human XVII type collagen provided by the invention has an amino acid sequence as shown in any one of SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 8. The invention also discloses a preparation method of the recombinant collagen and application of the recombinant collagen in preparation of anti-hair loss products. The recombinant human XVII type protein can effectively promote proliferation of human dermal papilla cells, increase expression of vascular endothelial growth factors and remarkably promote hair regeneration of unhaired mice, and has good application prospects of anti-hair loss products.

Description

Technical Field

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

[0002] Human type XVII collagen is an important transmembrane protein, mainly found in the hemidesmosomes of the epidermal basement membrane. It is a crucial component of the extracellular matrix, playing a vital role in maintaining the integrity of the dermal-epidermal junction and promoting the structural stability of the skin and hair follicles. Skin aging, whether natural or caused by ultraviolet radiation or environmental pollution, is associated with the loss of type XVII collagen. The function and structure of human type XVII collagen are related; its N-terminus is located in the cytoplasm, and its C-terminus extends into the extracellular matrix. It contains 16 non-collagenous domains (NC1-NC16) and 15 collagenous domains (C1-C15), with C15 being the largest collagenous domain. These domains are finely assembled into trimers, connecting the cytoskeleton and basement membrane, providing a niche for epidermal stem cells, and maintaining normal cellular function.

[0003] Recent studies have revealed high expression of type XVII collagen in hair follicle stem cells located in the bulge of the hair follicle. Melanocyte stem cells adhere directly to these stem cells and do not express type XVII collagen themselves. Loss of type XVII collagen triggers aging of hair follicle stem cells, affecting melanocyte adhesion and leading to gray hair. Simultaneously, the hair follicles themselves gradually atrophy, resulting in hair loss. Therefore, maintaining or supplementing type XVII collagen levels may be an effective means of preventing gray hair and hair loss, and its application in preventing hair loss and promoting hair growth has become a hot topic in recent years.

[0004] Type XVII collagen is present in very small amounts in the body, and its transmembrane structure means that traditional extraction methods cannot yield sufficient quantities, limiting early research and exploration of its functions and applications. With the development of synthetic biology, although recombinant expression of full-length type XVII collagen remains extremely difficult, expression of functional fragments has become a reality. Recombinant expression sequences are typically short fragments that are spliced ​​and / or repeated. Longer fragments are more difficult to express in existing expression systems such as *E. coli* and yeast, while shorter fragments may result in the loss of certain essential biological functions. Summary of the Invention

[0005] In view of the above-mentioned prior art, this application provides a new recombinant type XVII collagen, its preparation method and application.

[0006] Technical solution: This application expresses a natural long fragment from the C15 collagen domain of human type XVII collagen in Escherichia coli, and obtains recombinant human type XVII collagen with high soluble expression level.

[0007] The recombinant human type XVII collagen described in this application has an amino acid sequence as shown in any one of SEQ ID NO.1, SEQ ID NO.7, and SEQ ID NO.8.

[0008] The nucleic acid sequence encoding the protein shown in SEQ ID NO.1 is shown in SEQ ID NO.4, the nucleic acid sequence encoding the protein shown in SEQ ID NO.7 is shown in SEQ ID NO.9, and the nucleic acid sequence encoding the protein shown in SEQ ID NO.8 is shown in SEQ ID NO.10.

[0009] This application also provides a recombinant expression vector containing the above-mentioned nucleic acid. The expression vector is selected from pET22a, pET28a, pET32a, etc.

[0010] This application also provides an engineered bacterium or cell containing the above-mentioned expression vector.

[0011] The method for preparing recombinant type XVII collagen described in this application includes the following steps: (1) The coding genes are respectively ligated into expression vectors to obtain recombinant vectors; (2) Transform the recombinant vector obtained in step (1) into competent cells BL21(DE3) to obtain the recombinant strain; (3) After culturing the recombinant strain obtained in step (2), centrifuge to break the cells, resuspend in buffer, and then centrifuge again to collect the supernatant; (4) The supernatant obtained in step (3) is separated and purified to obtain recombinant human type XVII collagen.

[0012] Preferably, in step (1), the expression vector is derived from pET22a, pET28a, pET32a, etc., or is appropriately modified based on these vectors. The pET28a expression vector is further preferred.

[0013] Preferably, in step (2), the expression host is competent cells BL21(DE3).

[0014] This application also provides the application of the recombinant human type XVII collagen in the preparation of anti-hair loss products, pharmaceuticals, cosmetics, biomaterials, and medical devices. Specifically, the recombinant human type XVII collagen promotes the proliferation of NIH / 3T3 cells and human dermal papilla cells, and increases the expression of vascular endothelial growth factor.

[0015] The hair loss prevention products may include shampoos, conditioners, serums, sprays, essential oils, hair masks, massage creams, serum patches, solutions, foams, lotions, creams, injections, microneedles, etc.

[0016] Beneficial effects: This application expresses a natural long fragment from the C15 collagen domain of human type XVII collagen in Escherichia coli, obtaining recombinant human type XVII collagen with high soluble expression levels; the recombinant human type XVII protein can effectively promote the proliferation of human dermal papilla cells, increase the expression of vascular endothelial growth factor, and significantly promote hair regeneration in hair-depleted mice, showing great promise for use in anti-hair loss products. Attached Figure Description

[0017] Figure 1 These are SDS-PAGE images of SEQ ID NO. 1~3 expressed in shake flasks; Figure 2 These are SDS-PAGE images of fermenters containing SEQ ID NO.1 and SEQ ID NO.3. Figure 3 These are SDS-PAGE images of SEQ ID NO.1 and SEQ ID NO.7~8 expressed in shake flasks; Figure 4 It is the NIH / 3T3 proliferative activity of recombinant human type XVII collagen (SEQ ID NO.7); Figure 5 It is the human dermal papilla cell proliferation activity of recombinant human type XVII collagen (SEQ ID NO.7); Figure 6 It is the activity of SEQ ID NO.7 recombinant human type XVII collagen in promoting vascular endothelial growth factor; Figure 7 It is SEQ ID NO.7 Recombinant human type XVII collagen hair growth test in mice. Detailed Implementation

[0018] The technical solution of this application will be described in detail below with reference to specific embodiments.

[0019] Example 1: Sequence design and strain construction of recombinant type XVII collagen The specific sequence in this embodiment is derived from COHA1_HUMAN (UniProt Q9UMD9). Three single natural fragments of approximately 10kDa, 15kDa, and 20kDa were selected from the C15 domain, respectively. The N-terminus is attached with a short tag containing 6xHis. The amino acid sequences are shown in SEQ ID NO. 1~3, and the corresponding nucleic acid sequences after codon optimization are shown in SEQ ID NO. 4~6.

[0020] SEQ ID NO.1 MSKIKHHHHHHGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPG SEQ ID NO.2 MSKIKHHHHHHGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTG SEQ ID NO.3 MSKIKHHHHHHGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEG SEQ ID NO.4 ATGTCTAAAATAAAACACCATCATCATCATCATggtccacaaggaccaaagggtcaaaaaggcagcgtgggagatcctggcatggaaggccccatgggccagagagggcgagaaggccccatgggacctcgtggtgaggcagggcctcctggatctggagagaaaggggaaagaggggctgctggtgaaccaggtcctcatggcccacctggtgtcccaggttctgtgggtcccaaaggttccagcggctctcctggcccacagggccctccaggtcctgtaggtctccaagggctccgaggtgaagtaggacttcctggtgtcaaaggtgacaaaggaccaatgggaccaccaggaTAA SEQ ID NO.5 ATGTCTAAAATAAAACACCATCATCATCATCATggtccacaaggaccaaagggtcaaaaaggcagcgtgggagatcctggcatggaaggccccatgggccagagagggcgagaaggccccatgggacctcgtggtgaggcagggcctcctggatctggagagaaaggggaaagaggggctgctggtgaaccaggtcctcatggcccacctggtgtcccaggttctgtgggtcccaaaggttccagcggctctcctggcccacagggccctccaggtcctgtaggtctccaagggctccgaggtgaagtaggacttcctggtgtcaaaggtgacaaaggaccaatgggaccaccaggacccaaaggtgaccagggtgagaaaggacctcgaggcctcacaggcgagcctggcatgagaggtttgcctggtgctgttggtgagcccggggctaaaggagcaatgggtcctgctggcccagacggacaccaaggcccaagaggtgaacaaggtcttactgggTAA SEQ ID NO.6 ATGTCTAAAATAAAACACCATCATCATCATCATggtccacaaggaccaaagggtcaaaaaggcagcgtgggagatcctggcatggaaggccccatgggccagagagggcgagaaggccccatgggacctcgtggtgaggcagggcctcctggatctggagagaaaggggaaagaggggctgctggtgaaccaggtcctcatggcccacctggtgtcccaggttctgtgggtcccaaaggttccagcggctctcctggcccacagggccctccaggtcctgtaggtctccaagggctccgaggtgaagtaggacttcctggtgtcaaaggtgacaaaggaccaatgggaccaccaggacccaaaggtgaccagggtgagaaaggacctcgaggcctcacaggcgagcctggcatgagaggtttgcctggtgctgttggtgagcccggggctaaaggagcaatgggtcctgctggcccagacggacaccaaggcccaagaggtgaacaaggtcttactgggatgcctggaatccgtggcccaccaggaccttctggagacccaggaaagccaggtctcacaggaccccagggacctcagggacttcccggtacccctggccgaccaggaataaaaggtgaaccaggagctccaggcaagatcgtgacttcggaggggTAA Furthermore, natural short peptides derived from human type XVII collagen were selected and added to the N-terminus or both termini of the SEQ ID NO.1 sequence, and two amino acid sequences as shown in SEQ ID NO.7-8 were designed, and the corresponding nucleic acid sequences were as shown in SEQ ID NO.9-10.

[0021] SEQ ID NO.7 MSKIKGPRGHKGEKGDKGDQVYAGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGLKAEANGDLKTVSTKGKT SEQ ID NO.8 MSKIKGPRGHKGEKGDKGDQVYAGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPG SEQ ID NO.9 ATGTCTAAAATAAAAGGTCCGCGTGGCCATAAAGGCGAGAAAGGCGATAAAGGCGATCAGGTGTATGCGggtccacaaggaccaaagggtcaaaaaggcagcgtgggagatcctggcatggaaggccccatgggccagagagggcgagaaggccccatgggacctcgtggtgaggcagggcctcctggatctggagagaaaggggaaagaggggctgctggtgaaccaggtcctcatggcccacctggtgtcccaggttctgtgggtcccaaaggttccagcggctctcctggcccacagggccctccaggtcctgtaggtctccaagggctccgaggtgaagtaggacttcctggtgtcaaaggtgacaaaggaccaATGGGACCACCAGGACTGAAAGCGGAAGCGAACGGCGATCTGAAAACCGTGAGCACCAAAGGCAAAACCTAA SEQ ID NO.10 ATGTCTAAAATAAAAGGTCCGCGTGGCCATAAAGGCGAGAAAGGCGATAAAGGCGATCAGGTGTATGCGggtccacaaggaccaaagggtcaaaaaggcagcgtggggagatcctggcatggaaggccccatgggccagagagggcgagaaggccccatgggacctcgtggtgaggcagggcctcctggatctggagag aaaggggaaagaggggctgctggtgaaccaggtcctcatggcccacctggtgtcccaggttctgtgggtcccaaaggttccagcggctctcctggccca cagggccctccaggtcctgtaggtctccaagggctccgaggtgaagtaggacttcctggtgtcaaaggtgacaaaggaccaatgggaccaccaggaTAA After synthesizing the full gene of the nucleic acid sequence, it was constructed into the pET28a vector, transformed into the host BL21(DE3), plated on an antibiotic resistance selection plate, and positive clones were selected for PCR verification and gene sequencing to obtain the recombinant human type XVII collagen expression strain.

[0022] Example 2 Expression and purification of recombinant human type XVII collagen The recombinant human type XVII collagen expression strain was cultured in shake flasks using LB medium (5.0 g / L yeast extract, 10.0 g / L peptone, 10.0 g / L NaCl) for seed culture and fermentation. The growth temperature was 37℃, and the shaking speed was 200 rpm. 0.2 mM IPTG was used as the inducer, and the fermentation temperature after adding the inducer was either 16℃ or 37℃, with an induction time of 12 h. The seed culture medium for the recombinant human type XVII collagen expression strain in the fermenter was LB medium, and the fermentation medium was TB medium (24.0 g / L yeast extract, 12.0 g / L peptone, 0.4% (v / v) glycerol, 17 mM potassium dihydrogen phosphate, and 72 mM dipotassium hydrogen phosphate). The inoculum size was 5%. The initial fermentation conditions were: temperature 37 ± 0.5℃, aeration rate 0.8 VVM, rotation speed 200 rpm, tank pressure 0.03~0.04 MPa, pH 7.0. After adding the inducer, the fermentation temperature was 25 ± 0.5℃. The rotation speed, aeration rate, and tank pressure were adjusted to maintain dissolved oxygen at 10~30%, and the induction time was 12 h.

[0023] After high-speed centrifugation, the bacterial cells were collected, resuspended in phosphate buffer, then homogenized using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation for SDS-PAGE electrophoresis and purification.

[0024] The purification process was as follows: the liquid containing the target protein after fragmentation was precipitated and salted out with 0-30% ammonium sulfate, followed by Ni column affinity chromatography under 30 mM imidazole conditions, washing with impurities under 150 mM imidazole conditions, and eluting under 300 mM imidazole conditions. The sample purity could reach over 70%. To further improve the sample purity and remove impurities such as pigments, ion exchange chromatography was used for further purification. The equilibration conditions were 50 mM NaCl, pH 6.0, and the elution conditions were 200 mM NaCl, pH 6.0. The purified target protein solution was stored at low temperature for cell activity and efficacy testing.

[0025] The expression of recombinant human type XVII collagen is as follows: Figure 1-3 As shown. Figure 1 The image shows the SDS-PAGE of shake-flask expression of SEQ ID NO.1~3. Lane M is the marker. Lanes 1, 5, and 9 represent the soluble components of SEQ ID NO.1~3 induced at 37℃, lanes 2, 6, and 10 represent the inclusion body components of SEQ ID NO.1~3 induced at 37℃, lanes 3, 7, and 11 represent the soluble components of SEQ ID NO.1~3 induced at 16℃, and lanes 4, 8, and 12 represent the inclusion body components of SEQ ID NO.1~3 induced at 16℃. It can be seen that these three proteins are solublely expressed at both temperatures, and the expression level at 37℃ (indicated by the arrow) is higher than that at 16℃. The expression levels of SEQ ID NO.1 and SEQ ID NO.3 are slightly higher than those of SEQ ID NO.2 under the same conditions.

[0026] Figure 2 The image shows the SDS-PAGE of proteins of SEQ ID NO.1 and SEQ ID NO.3 expressed on a 10L fermenter. Lane M is the marker, lanes 1 and 3 are the soluble components of SEQ ID NO.1 and SEQ ID NO.3, respectively, and lanes 2 and 4 are the inclusion body components of SEQ ID NO.1 and SEQ ID NO.3, respectively. It can be seen that SEQ ID NO.1 is highly expressed on the fermenter, which is significantly higher than its expression level in shake flask and under the same conditions as SEQ ID NO.3.

[0027] Figure 3This is an SDS-PAGE image of shake-flask expression of SEQ ID NO.1 with a short peptide derived from type XVII collagen. Lane M is the marker, lanes 1, 3, and 5 are the soluble components of SEQ ID NO.1, SEQ ID NO.7, and SEQ ID NO.8 induced at 37℃, respectively, and lanes 2, 4, and 6 are the inclusion body components of SEQ ID NO.1, SEQ ID NO.7, and SEQ ID NO.8 induced at 37℃, respectively. It can be seen that adding the short peptide at the N-terminus or both ends significantly increased the expression level, approximately 1.5 times that of the SEQ ID NO.1 protein. The protein of SEQ ID NO.7 will be selected for further cell activity and efficacy studies.

[0028] Example 3: Proliferative Activity of Recombinant Type XVII Collagen in NIH / 3T3 Cells The recombinant human type XVII collagen from SEQ ID NO.7 prepared in this example was purified, and its cell proliferation activity was measured. NIH / 3T3 cells were seeded at a density of approximately 50% in 96-well plates, with 100 μL of cell suspension added to each well, and incubated overnight at 37°C. On day 2, purified samples were added in serially diluted amounts of 500 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL to wells containing NIH / 3T3 cell culture. Bovine type I collagen (batch number 380008-202001, 20 mg / vial, China National Institutes for Food and Drug Control) was added as a control. Each concentration was added in triplicate. After incubation at 37°C for 48 h, cell proliferation was detected using a CCK8 assay kit (A311-01, Vazyme), 10 μL per well. The absorbance was measured at 450 nm using an ELISA reader, which indirectly reflects the number of viable cells. The average values ​​were used to calculate the Cell viability plateau value and EC50 value. Cell viability is defined as the cell proliferation survival rate (i.e., fold increase) compared to the untreated control. EC50 refers to the drug concentration required for cells to proliferate to half of the highest plateau value.

[0029] Proliferating activity of NIH / 3T3 cells, such as Figure 4 As shown, recombinant human type XVII collagen has a significant pro-proliferative effect on NIH / 3T3 cells. Figure 2 The calculated Cell viability plateau value and EC50 value were 1.43 and 4.351 µg / mL, respectively. The Cell viability plateau value was higher than that of bovine type I collagen under the same conditions (1.21).

[0030] Example 4: Proliferative Activity of Recombinant XVII Collagen in Human Dermal Dermal Papillary Cells This embodiment measures the proliferative effect of recombinant human type XVII collagen from SEQ ID NO.7 at different concentrations on human dermal papillary cells (HDPCs).

[0031] Cell culture and drug administration: Human dermal papilla cells (HDPCs) in logarithmic growth phase (FH-Y052, Shanghai Fuheng Biotechnology Co., Ltd.) were cultured at a rate of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 10 μg / mL in 96-well plates. The plates were then incubated in a 5% CO2 incubator at 37°C. After 24 hours, the culture medium was aspirated, and the cells were washed with PBS. Serum-free medium containing the sample was then added to each well to create a blank control, experimental control, and zero-well control. The experimental control wells contained different concentration gradients of the active ingredient, corresponding to final concentrations of 200 μg / mL, 100 μg / mL, 20 μg / mL, and 4 μg / mL, respectively. Each control group was replicated in triplicate.

[0032] Cell viability assay: Add 10 μL of CCK8 to 100 μL of culture medium in each 96-well plate and incubate in a CO2 incubator in the dark for 1 h. The experimental procedure was performed according to the CCK8 kit (A311-01, Vazyme), and the absorbance at λ=450 nm was measured using a microplate reader.

[0033] Formula for calculating cell viability:

[0034] Experimental results are as follows Figure 5 As shown, incubating HDPCs with recombinant human type XVII collagen at concentrations of 20–200 μg / mL revealed that the proliferation-promoting effect of recombinant human type XVII collagen was concentration-dependent. Cell viability increased by 40% at 200 μg / mL compared to the control group, by 29% at 100 μg / mL, and by 19% at 20 μg / mL. This indicates that recombinant human type XVII collagen promoted the proliferation of HDPCs and enhanced their hair-inducing ability.

[0035] Example 5: Activity of recombinant type XVII collagen in promoting vascular endothelial growth factor This embodiment evaluates the effects of recombinant human type XVII collagen on hair root cell differentiation and improved blood circulation by detecting the effect of recombinant human type XVII collagen sample from SEQ ID NO.7 on the expression level of vascular endothelial growth factor (VEGF) in cells.

[0036] Cell culture and drug administration: HDPCs cells in logarithmic growth phase (FH-Y052, Shanghai Fuheng Biotechnology Co., Ltd.) were cultured at a concentration of 3 × 10⁻⁶ cells / cells. 5 The sample was seeded at a density of 2 mL per well in 6-well plates, divided into a blank control group and an experimental group, with 3 replicates in each group. The plates were cultured in DMEM complete medium until 70-80% confluence, the medium was removed, and the plates were washed with PBS. The test samples were then added to the plates at final concentrations of 100 μg / mL, 20 μg / mL, and 4 μg / mL, respectively, and cultured for another 24 h.

[0037] VEGF expression level detection: Discard the cell supernatant, lyse the cells with Trozol, and perform the operation according to the instructions of HiScript II OneStep qRT-PCR SYBR Green Kit (Q221-01, Vazyme). After RNA extraction, cDNA synthesis, RT-qPCR and other steps, the data were collected and the results were calculated.

[0038] Collect real-time PCR data. Use Ct as the amplification result, with the amplification amount of the GAPDH gene as the internal reference gene, and calculate the relative expression level of each gene (VEGF) as the test result.

[0039]

[0040] In the formula: ΔCt: The difference between the cycle number of the target gene and the cycle number of the internal reference gene; ΔCt: The difference between the average ΔCt of the test group and the average ΔCt of the blank control group; 2 -ΔΔCt : Relative expression level of the target gene in the blank control group / test group.

[0041] The experimental results are shown in Figure 6 As shown, compared with the untreated blank control group, recombinant human type XVII collagen at concentrations of 20 μg / mL and 100 μg / mL significantly increased the mRNA expression of vascular endothelial growth factor, indicating that recombinant human type XVII collagen can improve microcirculation, increase blood supply, and thus promote hair follicle growth.

[0042] Example 6 Hair Growth Test in C57BL / 6 Mice Promoting Hair Removal This embodiment evaluates the effect of recombinant human type XVII collagen on hair growth in hair-removed C57BL / 6 mice by detecting the effect of recombinant human type XVII collagen sample from SEQ ID NO.7 on hair growth on the back of hair-removed C57BL / 6 mice.

[0043] Experimental animals: SPF-grade male C57BL / 6 mice, 6-8 weeks old, purchased from Yangzhou University. Mice were housed individually in cages on a laminar flow rack in a clean animal husbandry facility, provided with ample food and water, and cared for by designated personnel. The ambient temperature was controlled at 18-22℃, relative humidity at 50-70%, with a 12h light / 12h dark cycle. Mice underwent acclimatization for one week before subsequent experiments.

[0044] Model establishment and grouping for drug administration: To synchronize the growth cycle of the hair on the backs of all mice, approximately 2×4 cm of the mouse backs were carefully trimmed using a shaver. 2 Hair was shaved from the shaved area, and then rosin and paraffin were melted in a 1:1 ratio. Mice were anesthetized and kept in a back-up position. A 2cm × 4cm piece of medical gauze soaked in rosin / paraffin was placed on the mouse's back and removed after cooling and solidification. The shaved mice were randomly divided into three groups (n=4 per group): a model group, a 2% minoxidil positive control group, and a recombinant type XVII collagen group. Drug intervention was administered 24 hours after modeling. The model group received no treatment, the minoxidil group received 2% minoxidil ointment, and the sample group received 100 μg / mL recombinant type XVII collagen, administered transdermally at a dose of 200 μL / mouse / day for two consecutive weeks. Hair growth in the shaved area was recorded during the experiment.

[0045] Experimental results: Hair growth on the backs of mice was as follows Figure 7 As shown, during hair growth in C57BL / 6 mice, the skin changed from pink to dark gray and then black. The skin at the hair removal site (day 1) in C57BL / 6 mice was light pink. On day 4 of administration, the backs of mice in the positive control group and the recombinant human type XVII collagen group began to turn light gray. On day 7 of administration, the backs of mice in all three groups turned grayish-black. On day 10 of administration, the backs of mice in the positive control group and the recombinant human type XVII collagen group showed centrally diffused growth of shiny black new hair. On day 14 of administration, the hair in the positive control group and the recombinant human type XVII collagen group was almost fully grown, while the hair in the negative control group was not fully grown. This indicates that recombinant human type XVII collagen has the effect of promoting hair growth in mice.

Claims

1. A recombinant human type XVII collagen, characterized in that, The amino acid sequence is shown in any one of SEQ ID NO.1, SEQ ID NO.7, or SEQ ID NO.

8.

2. The nucleic acid encoding the protein shown in SEQ ID NO.1, with the sequence shown in SEQ ID NO.4; the nucleic acid encoding the protein shown in SEQ ID NO.7, with the sequence shown in SEQ ID NO.9; and the nucleic acid encoding the protein shown in SEQ ID NO.8, with the sequence shown in SEQ ID NO.

10.

3. A recombinant expression vector containing the nucleic acid of claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The carrier is selected from pET22a, pET28a, and pET32a.

5. An engineered bacterium or cell containing the expression vector as described in claim 3 or 4.

6. The method for preparing recombinant human type XVII collagen according to claim 1, characterized in that, Includes the following steps: (1) The coding genes shown in claim 2 are respectively ligated into Escherichia coli expression vectors to obtain recombinant vectors; (2) Transform the recombinant vector obtained in step (1) into competent cells BL21(DE3) to obtain the recombinant strain; (3) After culturing the recombinant strain obtained in step (2), centrifuge to break the cells, resuspend in buffer, and then centrifuge again to collect the supernatant; (4) The supernatant obtained in step (3) is separated and purified to obtain recombinant human type XVII collagen.

7. The use of the recombinant human type XVII collagen as described in claim 1 in the preparation of hair loss prevention products, pharmaceuticals, cosmetics, biomaterials, and medical devices.

8. The application according to claim 7, characterized in that, The recombinant human type XVII collagen promotes the proliferation of NIH / 3T3 cells and human dermal papilla cells, and increases the expression of vascular endothelial growth factor.

9. The application according to claim 7, characterized in that, The hair loss prevention products are selected from shampoos, conditioners, serums, sprays, essential oils, hair masks, massage creams, serum patches, solutions, foams, lotions, creams, injections, and microneedles.

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