Recombinant collagen xvii for alleviating androgen-induced hair follicle damage, production method and use

Abstract

The present application belongs to the technical field of synthetic biology, and particularly relates to a recombinant collagen XVII for relieving androgen-induced hair follicle damage, a production method and application. The amino acid sequence of the recombinant collagen XVII is shown as SEQ ID NO. 1. The recombinant collagen XVII domain protein is easy to purify, has the functions of significantly relieving androgen-induced hair follicle damage, and promoting the proliferation and migration of human dermal hair papilla cells after androgen damage, and can be widely used in hair regeneration-related drugs, cosmetics or tissue engineering products.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to a recombinant type XVII collagen for alleviating androgen-induced hair follicle damage, its production method, and its application. Background Technology

[0002] Hair primarily serves to protect the scalp, shielding it from minor impacts and aiding in the evaporation of sweat. Hair loss (especially androgenetic alopecia) has become a prevalent public health problem worldwide, significantly impacting mental health and quality of life. Its pathogenesis involves multiple factors, including genetics, hormones, immunity, and stem cell / microenvironment degeneration. Currently, minoxidil and finasteride are effective medications for promoting hair growth in most patients with androgenetic alopecia. However, these medications may cause adverse side effects such as erectile dysfunction, birth defects, and hirsutism.

[0003] Collagen Type XVII, with a molecular weight of 180 kDa, is encoded by the COL17A1 gene. It is a rare type II transmembrane protein, primarily expressed by basal keratinocytes. Its most distinctive structural feature is its intracellular N-terminus, composed of 501 amino acids, containing four tandem repeat sequences of 24-26 amino acids each. The C-terminus is a relatively long extracellular polypeptide chain, part of the connection structure between the epidermis and dermis, mainly located in the basement membrane region. It forms anchoring plaques with proteins such as integrin α6β4, BP230, and laminin-332, connecting to the anchoring fibers of the lamina lucidum of the basement membrane. The fifteenth collagen domain is the longest collagen domain in type XVII collagen.

[0004] Type XVII collagen is a protein produced in the deepest layer of the epidermis. It is not only a structural protein but also participates in regulating the migration, division, and homeostasis of hair follicle stem cells. The depletion of type XVII collagen in hair follicle stem cells is a key factor leading to hair follicle atrophy and hair loss. Hair papilla cells are one of the earliest cell types identified as regulators of HFSCs. During hair follicle development, growth, cycle, and aging, they release various signals, activating signaling pathways such as Wnt and TGF-β, activating hair follicle stem cells, and promoting hair follicles to enter the anagen phase. They possess significant hair-promoting biological functions, thereby alleviating the damage caused by androgenetic alopecia.

[0005] With the large-scale application of genetic engineering technology, recombinant collagen expression has successfully solved the bottleneck of large-scale collagen production by using a modeled expression host to express exogenous proteins. Compared with traditional animal collagen extraction, recombinant collagen production has the following advantages: (1) It produces a wide variety of collagens, including common bovine, porcine, and fish collagens, as well as human collagens, which have better safety and immune efficacy in fields such as medical devices. (2) It can effectively avoid animal-derived diseases. Recombinant collagen usually uses simple prokaryotic or eukaryotic cells as expression hosts. Due to the huge difference in cell structure between recombinant collagen and human cells, the pathogens cannot be transmitted between them. (3) It has a short production cycle, saves costs, and can be rapidly scaled up, making it suitable for large-scale industrial production. Compared with animal culture, which takes 3 to 5 months, microbial culture only takes 2 to 3 days. It is simple to culture and can be carried out on a large scale with simple C and N sources, making it easy to prepare for industrial use. Therefore, the preparation of recombinant collagen, especially recombinant human collagen, is currently a hot topic in collagen production research.

[0006] Currently, the main problems in the production of type XVII collagen are as follows: 1) Natural type XVII collagen is difficult to obtain; 2) Recombinant type XVII collagen is difficult to express, and the expression level of full-length type XVII collagen in various existing expression systems is very low, which is difficult to meet the requirements of industrial production; 3) The biological activity of type XVII collagen fragments is unclear. Currently, it has only been reported that type XVII collagen is involved in the regulation of hair follicles in relation to the extracellular matrix, and there are almost no reports on the biological activity of type XVII collagen fragments.

[0007] Based on the above problems, how to obtain type XVII collagen fragments so that they can be efficiently expressed in microbial expression systems (especially secreted expression in Pichia pastoris) and have clear and prominent biological activities has always been a technical problem in this field. Summary of the Invention

[0008] In view of the technical problems existing in the prior art, the purpose of the present invention is to provide a new recombinant type XVII collagen, which can be efficiently secreted and expressed in a commercial Pichia pastoris expression system and has good biological activity in promoting the migration and proliferation of human dermal papilla cells.

[0009] The technical solution of the present invention includes:

[0010] In a first aspect, the present invention provides a recombinant type XVII collagen for alleviating androgen-induced hair follicle damage, which is a truncated protein of human type XVII collagen, and its amino acid sequence is shown in SEQ ID NO.1 (i.e., positions 604 to 809 in the COL17A1 sequence).

[0011] In a second aspect, the present invention provides a nucleic acid encoding the recombinant type XVII collagen, the sequence of which is shown in SEQ ID NO.2.

[0012] In a third aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0013] In a fourth aspect, the present invention provides a host cell in which the expression vector is introduced.

[0014] In a preferred embodiment of the present invention, the host cell is a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, and Bacillus licheniformis, and eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells.

[0015] More preferably, the host cell is Escherichia coli or Pichia pastoris.

[0016] More preferably, the host cell is Pichia pastoris.

[0017] In a fifth aspect, the present invention provides a method for producing the recombinant type XVII collagen, comprising the steps of culturing the host cells to express the recombinant type XVII collagen and collecting the culture containing the recombinant type XVII collagen.

[0018] In a preferred embodiment of the present invention, the expression is one or a combination of two of the following: constitutive expression and induced expression.

[0019] More preferably, the inducer for induced expression is an inducer such as IPTG, β-galactoside, methanol, or ethanol.

[0020] In a preferred embodiment of the present invention, the production method further includes a step of separating and purifying the culture.

[0021] More preferably, the separation and purification employs one or more of the following methods in combination: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation.

[0022] Furthermore, the separation and purification employs a combination of chromatographic chromatography and membrane separation or a combination of ion exchange chromatography and membrane separation.

[0023] In a sixth aspect, the present invention provides the use of the recombinant type XVII collagen in the preparation of products for promoting hair follicle cell proliferation or hair regeneration.

[0024] As a preferred embodiment of the present invention, the product is used to alleviate hair follicle damage caused by androgens.

[0025] In a preferred embodiment of the present invention, the product is a pharmaceutical, cosmetic, tissue engineering material, or medical device.

[0026] In a seventh aspect, the present invention provides a product for promoting hair follicle cell proliferation or hair regeneration, comprising the recombinant type XVII collagen and acceptable excipients.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention provides a recombinant type XVII collagen that can be efficiently secreted and expressed in a commercial Pichia pastoris expression system. The recombinant type XVII collagen domain protein is easy to purify and has the functions of significantly alleviating androgen-induced hair follicle damage, promoting the proliferation and migration of human dermal papilla cells after androgen damage, promoting hair regeneration, and alleviating androgenetic alopecia. It can be widely used in hair regeneration-related pharmaceuticals, cosmetics, or tissue-engineered products. Attached Figure Description

[0029] Figure 1 This is a diagram of the plasmid vector used in the construction of the Pichia pastoris engineered strain expressing recombinant type XVII collagen, as shown in Example 1.

[0030] Figure 2 This is an SDS-PAGE analysis image of purified recombinant type XVII collagen. 1. Stock solution; 2. Flow-through; 3. Elution; 4. Elution; 5. Elution containing target protein; 6. Elution containing target protein.

[0031] Figure 3 This is a bar chart showing the cell proliferation of recombinant type XVII collagen.

[0032] Figure 4 This is a cell migration graph of recombinant type XVII collagen after 18 hours of culture. A, 0h experimental group; B, 18h experimental group; C, 0h blank control group; D, 18h blank control group; E, 18h cell migration bar graph.

[0033] Figure 5 HE staining images of animal experiments involving recombinant type XVII collagen. A) Blank group, B) Modeling group, C) Minoxidil group, D) Collagen group. Detailed Implementation

[0034] The specific embodiments of the present invention are described below with reference to the accompanying drawings. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on the technical concept of the present invention and existing technologies, and are not limited to the specific descriptions of the embodiments of the present invention.

[0035] Unless otherwise specified, the methods used in the embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0036] The definitions of terms used herein are intended to incorporate the generally accepted prior art definitions of each term in the field of biotechnology. Examples are provided where appropriate. Unless otherwise limited, either individually or as part of a larger group, these definitions apply to the terminology used throughout this specification.

[0037] This invention discovers that a portion of the fifteenth collagen domain from the α chain of natural human type XVII collagen can be efficiently secreted and expressed in a commercial Pichia pastoris expression system, and exhibits good biological activities in promoting human dermal papilla cell migration and cell proliferation.

[0038] Example 1

[0039] Preparation of recombinant type XVII collagen (XVII-COL15) in a Pichia pastoris expression system

[0040] I. Experimental Methods

[0041] 1. Preparation of shuttle plasmid and Pichia pastoris expression strain

[0042] According to the amino acid sequence shown in SEQ ID NO.1, codon optimization was performed on the Pichia pastoris expression system without changing the amino acid sequence to obtain the target gene sequence of recombinant type XVII collagen (XVII-COL15), and a new gene was introduced at the 5' end of the sequence. EcoR I restriction site and signal peptide cleavage site are introduced at the 3' end Not The optimized gene sequence was synthesized by cutting the I restriction site and then ligated into the expression vector pPIC9K to obtain the pPIC9K-XVII-COL15 plasmid.

[0043] The amino acid sequence of recombinant type XVII collagen (XVII-COL15) shown in SEQ ID NO.1 is as follows:

[0044] GREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKI

[0045] Nucleotide sequence of SEQ ID NO.2 recombinant type XVII collagen (XVII-COL15):

[0046] .

[0047] Using Pichia pastoris GS115 as the expression host, the pPIC9K-XVII-COL15 plasmid was linearized and then electroporated into Pichia pastoris GS115 competent cells. Electroporation was performed using an electroporator with the following parameters: voltage 1.5 kV, resistance 200 Ω, capacitance 25 μF, and electroporation time 5 ms. High copy selection was performed using the G418 gradient screening method to select the optimal transformant. The transformed strain was cultured at 30℃ for 72 h to obtain the Pichia pastoris genetically engineered strain, which is the Pichia pastoris expression strain.

[0048] 2. Induced expression of the target protein

[0049] (1) Select a single colony of the constructed Pichia pastoris expression strain and add it to 25ml of BMGY liquid medium (BMGY liquid medium contains 1% yeast extract, 10% PPB, 1.34% YNB, 2% peptone and 1% glycerol), and incubate at 30℃ and 220rpm for 24h (OD is 10~20) for activation;

[0050] (2) Transfer 1% of the inoculum to a 1000ml Erlenmeyer flask (containing 500ml of BMGY culture medium), and incubate at 30℃ and 220rpm for 24h (OD is 8~10) as the seed for the upper tank;

[0051] (3) Prepare a high-density fermentation medium (3L). The composition of the medium is as follows:

[0052] 120g glycerol, 27.3g K2SO4, 1.38g CaSO4·2H2O, 6.3g KOH, 22.5g MgSO4·7H2O, 40.2ml phosphoric acid, 2ml defoamer, 2500ml water.

[0053] Add the 3L culture medium to a 5L fermenter, sterilize at 121℃ for 20min, and after cooling to 30℃, add 12ml of trace elements. Before inoculation, adjust the pH to 5 with NH3·H2O. Add the seeds prepared in (2) to the fermenter in the form of flame inoculation for fermentation culture.

[0054] (4) When the OD reaches 150-200, stop feeding glycerol. After 30 minutes, measure the OD until it remains basically unchanged. Then, periodically feed methanol, adjusting the amount according to the dissolved oxygen level. Measure the OD every hour until it starts to decrease, at which point the bacteria can be harvested.

[0055] 3. Collagen purification

[0056] The protein supernatant collected by centrifugation was filtered through a hollow fiber column and then concentrated to 20% of its initial volume by ultrafiltration through a 5 kDa ultrafiltration membrane.

[0057] Equilibration buffers were prepared based on the isoelectric point of recombinant type XVII collagen: 20 mmol / L sodium phosphate buffer (Solution A, pH 6.0), with 20 mmol / L sodium phosphate buffer + 1.0 mol / L NaCl (Solution B, pH 6.0) as the elution buffer; the protein fermentation broth concentrated by ultrafiltration in the previous step was used to prepare the loading solution, which was filtered through a 0.22 μm filter membrane and loaded onto an MMC Large Scale chromatography column; the column was equilibrated with equilibration buffer before loading; after loading, the column was first washed with Solution A until the UV drop was gradual, and then gradient elution was performed with Solutions A and B at a flow rate of 10 mL / min; each eluted fraction was collected and analyzed by SDS-PAGE.

[0058] The protein was concentrated again using an ultrafiltration membrane with a molecular weight of 5 kDa to remove salt. When the conductivity of the protein solution dropped below 300 uS / cm, the protein was collected and analyzed by SDS-PAGE. Lyophilization yielded recombinant type XVII collagen.

[0059] Example 2

[0060] Biological function evaluation of recombinant type XVII collagen (XVII-COL15)

[0061] 1. HDPC cell experiments

[0062] This embodiment uses the recombinant type XVII collagen fragment (XVII-COL15) prepared in Example 1 to conduct a dihydrotestosterone (DHT)-induced damage experiment on human dermal dermal papilla cells (HDPCs) to evaluate the proliferative effect of XVII-COL15 on damaged HDPCs. The specific experimental method is as follows:

[0063] Human dermal dermal papilla cells that have grown to 70%–80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin containing EDTA and cultured in complete medium to achieve a cell density of 5 × 10⁻⁶ cells / mL. 4 Cell suspension at 100 μL / mL. Seed 100 μL of cell suspension into 96-well culture plates and incubate at 37°C with 5% CO2 and saturated humidity. After 24 h of cell culture, aspirate the complete culture medium.

[0064] Except for the blank control group, 20 μM DHT working solution was added to each well for modeling. After 24 hours of modeling, the working solution was precipitated. Different concentrations of recombinant type XVII collagen solution (0.5, 1, 3, 5 mg / ml, with 5 replicates per concentration) diluted in human dermal papilla cell culture medium were added to the experimental groups. The control group consisted of cells cultured in complete human dermal papilla cell culture medium, and the blank group consisted of cell-free human dermal papilla cell culture medium. The cells were cultured at 37℃ and 5% CO2 saturated humidity for 24 hours. After aspirating the culture medium from each group, 110 μL of a mixture of human dermal papilla cell culture medium and CCK-8 (100 μL of culture medium, 10 μL of CCK-8) was added and incubated in a cell culture incubator for 1–4 hours. The absorbance (OD value) of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The cell proliferation rate under DHT-induced damage was calculated based on the mean absorbance of each group using the following formula:

[0065] .

[0066] Experimental results are as follows Figure 3As shown, compared with the blank control group (100%), cells treated with DHT showed a significant inhibitory effect. The cells in the experimental group prepared by this invention exhibited a trend of first promoting and then inhibiting the proliferation of damaged cells with increasing DHT concentration. The optimal proliferative effect on human dermal papilla cells inhibited by DHT was observed at a protein concentration of 3 mg / ml, with a cell proliferation rate of 100.8 ± 5.0%, close to that of the blank control group (100%). This indicates that the recombinant type XVII collagen prepared by this invention can promote the proliferation of human dermal papilla cells after DHT damage and alleviate the damage caused by DHT.

[0067] 2. Cell migration experiment

[0068] Human dermal dermal papilla cells that have grown to 70%–80% of the culture dish area were harvested, digested with 0.25% trypsin, and cultured in complete medium to achieve a cell density of 2.5 × 10⁻⁶ cells / mL. 5 Cell suspension was prepared at 1 / mL. Using a marker and a ruler, three horizontal lines were drawn at the bottom of each well. 2 mL of the cell suspension was seeded into each 6-well plate and incubated at 37°C with 5% CO2 saturated humidity. After 24 h of cell culture, a 10 μl pipette tip was used to gently push downwards along the wells to create a longitudinal scratch. The culture medium was then aspirated, and the cells were washed three times with PBS to remove any scraped cells. In the experimental group, a 3 mg / mL solution of recombinant type XVII collagen prepared in Example 1 (diluted with serum-free human dermal papilla culture medium) was added (two replicates). The blank control group consisted of human dermal papilla serum-free culture medium without recombinant type XVII collagen added to the cells. Cells were incubated at 37°C with 5% CO2 saturated humidity for 18 h. The images were taken under a 10x microscope, focusing on the intersection of the horizontal and vertical streaks.

[0069] The results are as follows Figure 4 As shown, at 0h ( Figure 4 (As shown in A and C) No cells were found in the central scratch area of ​​both the blank control group and the experimental group. After 18 hours of incubation, all cells were cultured in an incubator. Figure 4 As shown in Figures B and D, the migration rate of human dermal papilla cells cultured in recombinant type XVII collagen solution (3 mg / ml) was 64.0 ± 2.8%, while the migration rate of cells cultured in serum-free human dermal papilla culture medium was only 35.8 ± 2.6%, showing a significant increase in cell migration rate. This indicates that recombinant type XVII collagen has the ability to promote the migration of human dermal papilla cells, thereby improving the hair follicle microenvironment and alleviating hair follicle damage caused by androgens.

[0070] 3. Animal experiments

[0071] Mice (n=40) were randomly divided into four groups after one week of acclimatization: a normal control group (saline, n=10), a positive control group (n=10, minoxidil tincture 20 mg / kg / day, applied topically, a drug commonly used to treat male pattern baldness and alopecia areata), a model group (n=10, testosterone propionate 5 mg / ml, injected), and a recombinant XVII collagen group (3 mg / ml, n=10, applied topically). On the first day of the experiment, a 2cm×2cm area on the back of the mice was shaved and depilatory cream was applied. Subsequently, the normal control group was injected with the same dose of saline, while the model group, positive control group, and recombinant XVII collagen group were injected with 5 mg / ml testosterone propionate daily. Half an hour after injection, the positive control group and recombinant XVII collagen group were respectively treated with 150 μl of minoxidil tincture and recombinant XVII collagen solution. Hair growth on the back of the mice was observed daily, and photographs were taken of the back skin of the mice every 3-5 days. On day 15 of the experiment, mice were photographed and euthanized by cervical dislocation. After euthanasia, the thymus, spleen, small intestine, colon, and liver were collected, along with skin from the back of the mice. All tissues were immediately fixed in 4% neutral formalin solution for 24 hours. Following fixation, the tissues were dehydrated using a gradient of ethanol, cleared with xylene, and embedded in paraffin to prepare continuous tissue sections approximately 5 μm thick. The tissue sections were then stained with hematoxylin and eosin (HE).

[0072] HE staining results of skin tissue from the hairless area on the back of mice are as follows: Figure 5 As shown, the blank control group ( Figure 5 Figure A shows the hair follicle condition of normal mice; the follicles are structurally intact and morphologically healthy. Hair follicles extend deep into the skin, are of normal number, and exhibit a natural growth cycle, serving as a baseline reference for the experiment. The modeling group (… Figure 5 Figure B shows the hair follicle condition in mice after androgen-induced hair follicle damage, with significantly inhibited hair follicle growth. This is manifested by a significant reduction in the number of hair follicles, with remaining follicles generally atrophied, shortened, and structurally indistinct, with shallower depth, successfully mimicking the pathological state of hair degeneration or growth arrest. Minoxidil group ( Figure 5 (Figure C) shows the condition of hair follicles after treatment with minoxidil following androgen damage, compared with the modeling group ( Figure 5 Compared to the control group (shown in Figure B), the treatment showed some therapeutic effect. The number of hair follicles increased, the atrophy was partially improved, and some signs of growth appeared, indicating that minoxidil has a promoting effect on hair follicle growth. However, its effect has not yet recovered to that of the control group (shown in Figure B). Figure 5 The health level of the collagen group (as shown in Figure A). Figure 5 (Figure D) shows the condition of hair follicles treated with recombinant type XVII collagen from Example 1 after androgen damage, with the most significant improvement in hair follicle growth. The number of hair follicles was significantly better than in the modeling group (Figure D). Figure 5 (as shown in B) and minoxidil group ( Figure 5 As shown in Figure C), more well-shaped hair follicles appeared, and its recovery level was closest to that of the normal control group ( Figure 5 (As shown in Figure A). The HE staining results clearly show that the hair follicle growth status is: collagen group > minoxidil group > modeling group. This indicates that recombinant type XVII collagen can alleviate hair loss caused by androgen-induced hair follicle damage.

[0073] Based on the above, the recombinant type XVII collagen expressed in this invention has good safety, can promote the proliferation of human dermal papilla cells under DHT damage, regulate the hair follicle microenvironment and thus regulate the hair follicle cycle and promote hair growth, and can also indirectly regulate the activity of hair follicle stem cells, thereby achieving the purpose of regulating intercellular signal transduction and promoting hair follicle proliferation. It has a good effect on promoting hair follicle cell proliferation and hair regeneration, and can be used to alleviate the damage of androgen-induced hair loss.

[0074] This invention is not limited to the embodiments described above. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of this invention. The scope of this invention is defined by the claims and their equivalents.

Claims

1. A recombinant type XVII collagen for alleviating androgen-induced hair follicle damage, characterized in that, The amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO.

1.

2. A nucleic acid encoding the recombinant type XVII collagen of claim 1, characterized in that, The sequence of the nucleic acid is shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, It contains the nucleic acid as described in claim 2.

4. A host cell incorporating the expression vector of claim 3, characterized in that, The host cell is Escherichia coli, Pichia pastoris, or Bacillus subtilis.

5. The host cell according to claim 4, characterized in that, The host cell is Pichia pastoris.

6. A method for producing recombinant type XVII collagen as described in claim 1, characterized in that, The method includes the steps of culturing host cells in which the recombinant type XVII collagen is introduced, enabling them to express the recombinant type XVII collagen, and collecting cultures containing the recombinant type XVII collagen.

7. The production method according to claim 6, characterized in that, It also includes the step of separating and purifying the culture.

8. The production method according to claim 7, characterized in that, The separation and purification process employs one or more of the following methods in combination: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation.

9. The use of the recombinant type XVII collagen according to claim 1 in the preparation of products for promoting hair follicle cell proliferation or hair regeneration, characterized in that, The product is used to alleviate hair follicle damage caused by androgens.

10. A product for promoting hair follicle cell proliferation or hair regeneration, characterized in that, It comprises the recombinant type XVII collagen as described in claim 1 and acceptable excipients.

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