Recombinant humanized collagen type xvn and preparation and application thereof

Abstract

This invention provides two novel recombinant humanized type XVII collagen proteins, whose amino acid sequences are shown in A and B, respectively. This invention also covers derivative products that retain collagen activity after modification such as substitution, insertion, deletion, or addition based on the said sequences, as well as functional variants with more than 80% homology to the said sequences. The collagen proteins consist of, or contain, the amino acid sequences shown in (A)n and / or (B)n, where n is an integer ≥1. The recombinant humanized type XVII collagen proteins of this invention can be efficiently expressed in a Pichia pastoris expression system, showing promising prospects for industrial production. Functional verification experiments show that this collagen protein possesses both excellent antioxidant activity and barrier repair function, and is expected to be widely used as a functional ingredient in medical repair and high-end cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant humanized type XVII collagen, its encoding nucleic acid molecule, expression vector, host cell, preparation method, and its application in the medical and cosmetic fields. Background Technology

[0002] Type XVII collagen (COL17), also known as BP180 / BPAG2, is a transmembrane protein widely expressed in various tissues such as skin, colon, esophagus, and placenta. It is a key regulator of skin aging and wound repair, playing a crucial role in maintaining youthful skin, protecting the activity of hair follicle stem cells, and promoting wound healing. Simultaneously, its dysfunction is closely related to hair loss, autoimmune bullous diseases, and the development of various tumors. Due to its multidimensional physiological regulatory functions, type XVII collagen has become a hot target in the fields of skin anti-aging, hair regeneration, and wound repair, possessing extremely high translational potential and market value in the pharmaceutical, health, and cosmetic industries.

[0003] However, type XVII collagen is extremely rare in human and animal tissues, and cannot be extracted in large quantities by traditional acid, alkali, and enzymatic hydrolysis methods. Furthermore, animal-derived collagen poses immunogenicity and potential biosafety risks such as viruses and diseases.

[0004] The emergence of recombinant humanized collagen technology provides a feasible solution to the aforementioned problems. Compared with animal-derived collagen, recombinant humanized collagen not only has better bioactivity and biocompatibility, but also lower pathogen risk and is easier for large-scale production. Currently, common recombinant expression systems include mammalian and insect cell expression systems, which are costly and have low yields; prokaryotic (E. coli) systems express recombinant proteins without post-translational modifications, and intracellular expression requires cell lysis, which can introduce pyrogens such as endotoxins. Pichia pastoris has been recognized as a Generally Recognized As Safe (GRAS) strain by the U.S. Food and Drug Administration (FDA), exhibiting good biosafety; simultaneously, this expression system has technical advantages such as high-density fermentation, high expression levels of exogenous proteins, extracellular secretion of recombinant proteins, post-translational modification capabilities for eukaryotic proteins, and low culture costs. Therefore, Pichia pastoris is the preferred expression host for the production of recombinant humanized type XVII collagen in this invention.

[0005] In view of this, the present invention provides two novel recombinant humanized type XVII collagen proteins with amino acid sequences of SEQ ID NO. 1 and SEQ ID NO. 2, respectively. They can be efficiently secreted and expressed by Pichia pastoris SMD1168 strain and have been experimentally verified to have excellent antioxidant and barrier repair functions. Summary of the Invention

[0006] The primary objective of this invention is to provide two novel recombinant humanized type XVII collagens.

[0007] A second object of the present invention is to provide a nucleic acid molecule encoding the recombinant humanized type XVII collagen.

[0008] A third objective of the present invention is to provide a recombinant expression vector containing the nucleic acid molecule.

[0009] A fourth objective of the present invention is to provide a host cell containing the nucleic acid molecule or recombinant expression vector.

[0010] The fifth objective of this invention is to provide a method for preparing the recombinant humanized type XVII collagen.

[0011] The sixth objective of this invention is to provide the application of the recombinant humanized type XVII collagen. Technical solution

[0012] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a recombinant humanized type XVII collagen, wherein the amino acid sequence of the collagen is as shown in (A) or (B) below: (A) The amino acid sequence as shown in SEQ ID NO.1; (B) The amino acid sequence as shown in SEQ ID NO.2.

[0013] As a preferred embodiment of the present invention, the collagen further comprises a functional variant having ≥80% sequence homology with the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the functional variant retains the antioxidant activity and / or barrier repair activity of the collagen.

[0014] As a preferred embodiment of the present invention, the amino acid sequence of the collagen is composed of repeating units as shown in (A)n and / or (B)n, or contains the repeating units; wherein n is an integer ≥1.

[0015] In a preferred embodiment of the present invention, n is an integer from 1 to 10, preferably an integer from 1 to 5.

[0016] As a preferred embodiment of the present invention, the amino acid sequence further includes a derived sequence obtained by substitution, deletion, insertion or addition of one or more amino acid residues based on the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the derived sequence has collagen biological activity.

[0017] In a second aspect, the present invention provides a nucleic acid molecule encoding the recombinant humanized type XVII collagen described in the first aspect.

[0018] Thirdly, the present invention provides a recombinant expression vector containing the nucleic acid molecules described in the second aspect.

[0019] Fourthly, the present invention provides a host cell containing the nucleic acid molecule described in the second aspect or the recombinant expression vector described in the third aspect, wherein the host cell is Escherichia coli, Pichia pastoris, or other recombinant protein expression system host cells.

[0020] Fifthly, the present invention provides a method for preparing recombinant humanized type XVII collagen, comprising the following steps: (1) Construct an expression vector containing a nucleic acid molecule encoding the recombinant humanized type XVII collagen described in the first aspect; (2) Transform the expression vector described in step (1) into host cells; (3) Inducing the expression of recombinant humanized type XVII collagen in the host cells described in step (2); (4) Collect and purify the expressed recombinant humanized type XVII collagen.

[0021] In a sixth aspect, the present invention provides the use of the recombinant humanized type XVII collagen described in the first aspect or the recombinant humanized type XVII collagen prepared by the method described in the fifth aspect in the preparation of products with antioxidant function and / or skin barrier repair function.

[0022] As a preferred embodiment of the present invention, the product is a skin care product, cosmetic, medical dressing, or pharmaceutical preparation.

[0023] As a preferred embodiment of the present invention, the application includes its use in the preparation of products for treating or preventing skin photoaging, promoting skin wound healing, and improving skin barrier function.

[0024] As a preferred embodiment of the present invention, the application includes its use in the preparation of products for the prevention or treatment of hair loss and for promoting the maintenance of hair follicle stem cells.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) The recombinant humanized type XVII collagen provided by the present invention is designed based on the functional region of human type XVII collagen, has high sequence identity with human collagen, has no risk of rejection or sensitization, and has high biosafety.

[0026] (2) The recombinant humanized type XVII collagen provided by the present invention has good biological activity. Experimental verification shows that it has excellent antioxidant activity and skin barrier repair function, and can promote cell proliferation, cell adhesion and cell migration.

[0027] (3) The recombinant humanized type XVII collagen provided by the present invention has good water solubility, high stability, and is easy to purify. It is suitable for large-scale industrial production and has broad application prospects in the fields of medical repair and cosmetics. Attached Figure Description

[0028] Figure 1 SDS-PAGE image of recombinant humanized type XVII collagen expression in Pichia pastoris SMD1168; Figure 2 Evaluation of the cytotoxicity of recombinant humanized type XVII collagen-1 to HaCaT cells; Figure 3 Evaluation of the cytotoxicity of recombinant humanized type XVII collagen-2 to HaCaT cells; Figure 4 : Curve of recombinant humanized type XVII collagen-1 promoting HaCat cell proliferation; Figure 5 : Curve of recombinant humanized type XVII collagen-2 promoting HaCat cell proliferation; Figure 6 : Recombinant humanized type XVII collagen promotes HaCat cell migration (Figure); Figure 7 Antioxidant activity of recombinant humanized type XVII collagen at the highest concentration; Figure 8 : Antioxidant activity curve of recombinant humanized type XVII collagen. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Construction, screening and recombinant expression of recombinant collagen-producing yeast strains Based on the full-length amino acid sequence of human type XVII collagen (UniProt ID: Q9UMD9), regions with high hydrophilicity, high antigenic index, and functional activity were screened through bioinformatics analysis. The amino acid sequences selected in this invention, SEQ ID NO.1 and SEQ ID NO.2, are truncated sequences from the full-length human type XVII collagen sequence. Considering the codon bias of Pichia pastoris, the nucleotide sequence was codon optimized, resulting in SEQ ID NO.3 and SEQ ID NO.4. The Pichia pastoris secretory expression vector pPIC9K was selected as the recombinant expression vector, with the gene insertion site between the signal cleavage site and NotI in the vector. The vector was then transformed into yeast SMD1168 cells for expression.

[0031] The pPIC9K-COL17 recombinant expression vector plasmid was linearized by digestion with SacI enzyme at 37°C for 30 min, and then the linearized plasmid was recovered using a PCR product purification kit.

[0032] The linearized plasmid was chemically transformed into Pichia pastoris SMD1168 competent cells. An appropriate amount of the transformed bacterial culture was spread on MD plates and incubated upside down at 30°C for 2-5 days until single colonies appeared.

[0033] Add 3 mL of sterile double-distilled water to the surface of an MD plate. Gently scrape off the transformants from the plate surface using a sterile spreader. Dilute the bacterial suspension with sterile water and spread an appropriate amount onto a YPD plate containing 0.5 mg / mL G418. Incubate at 30°C for 3–4 days until single colonies appear. Pick colonies from the YPD plate and spot them onto YPD plates containing 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL G418, respectively. Continue incubation at 30°C for 96–120 hours. Select transformants that grow on plates containing 4 mg / mL G418 as the recombinant yeast strain for trial expression. Then, select the strain with the highest yield for subsequent fermentation experiments.

[0034] Example 2: Fermentation expression of recombinant collagen The seed culture was inoculated into BMGY medium and cultured with shaking at 30℃ and 240 rpm for 24 h to allow for cell proliferation. The cell density OD was then measured. 600 Approximately 8-10 μL of bacterial culture was collected and resuspended in BMMY medium. Methanol was added to induce fermentation, with 0.5% methanol added every 24 hours. The culture was incubated at 27.5°C and 240 rpm with shaking for 72 hours. The supernatant of the fermentation broth was collected.

[0035] Example 3: Isolation and purification of recombinant collagen fermentation broth The supernatant was concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The supernatant was then pumped into the ultrafiltration system and concentrated 8 times. The concentrate was collected and filtered through a 0.45 μm membrane for later use.

[0036] Cation chromatography: Equilibrate the cation chromatography medium with equilibration buffer (20 mM tartaric acid, 100 mM NaCl, pH 4.0). After sample loading, elute with elution buffer (20 mM tartaric acid, 500 mM NaCl, pH 4.0) and collect the eluent. Dialyze using a 3KD concentration bag at 4°C, changing the dialysis buffer every 2–4 hours. The total dialysis time should be maintained at over 24 hours to ensure adequate dialysis.

[0037] The purified protein solution was concentrated by ultrafiltration and then lyophilized to obtain recombinant humanized type XVII collagen lyophilized product. The final purified recombinant collagen sample is shown below. Figure 1 .

[0038] Example 4: Evaluation of the cytotoxicity of recombinant humanized type XVII collagen to HaCaT cells Cell resuscitation and expansion: HaCaT cells were resuscitated and cultured in DMEM medium containing 10% FBS and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator. Cells were passaged periodically, and cells in the logarithmic growth phase were used for experiments.

[0039] CCK-8 toxicity test: Cells were seeded at a density of 5 × 10³ cells / well in 96-well plates, with 100 μL of culture medium per well. After 24 hours of culture and adherence, the medium was replaced with different concentrations of the sample, with three replicates for each concentration. Blank wells (culture medium only) and a solvent control group (PBS at the same volume as the highest concentration) were also included. After 24 hours of culture, 10 μL of CCK-8 solution was added to each well, and incubation continued for 1–4 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader.

[0040] Data analysis: Cell viability (%) = [(OD sample - OD blank) / (OD solvent control - OD blank)] × 100%. Plot dose-toxicity curves and calculate IC0 (non-toxic concentration) and IC50. 50 (50% inhibition concentration).

[0041] The results are as follows Figure 2 and Figure 3 As shown: Recombinant humanized type XVII collagen-1 showed no significant cytotoxicity at concentrations below 25 μg / mL, IC50... 50 ≈ 43 μg / mL; recombinant humanized type XVII collagen-2 showed no significant cytotoxicity at concentrations below 50 μg / mL, IC50...50 ≈ 93 μg / mL.

[0042] Example 5: Detection of the barrier repair function of recombinant humanized type XVII collagen (1) Experiment on the proliferation of HaCaT cells promoted by recombinant humanized type XVII collagen (cell kinetics level - barrier repair basis) Cell seeding: Digest, resuspend, and count well-grown HaCaT cells. Adjust the cell density to 5 × 10⁶ cells / year using complete culture medium. 4 cells / mL. Add 100 μL of cell suspension to each well of a 96-well plate (i.e., 5000 cells per well). Fill the edge wells with PBS to reduce errors caused by evaporation. Incubate at 37°C, 5% CO2 for 24 hours to allow the cells to adhere completely.

[0043] Change medium and add drugs: 24 hours later, carefully aspirate the old culture medium from the wells using a pipette. Add 100 μL of fresh complete culture medium containing the corresponding sample (solvent, positive control, sample).

[0044] Culture: Return the cells to the incubator and continue culturing for 48 hours (or 72 hours).

[0045] CCK-8 assay: Add 10 μL of CCK-8 solution directly to each well. Gently shake the plate to mix. Incubate for 1–4 hours (observe under a microscope during this time; the optimal detection time is when the solution in the solvent control group turns orange-red and the OD value is between 0.8 and 1.2). Measure the absorbance (OD value) of each well using a microplate reader at a wavelength of 450 nm.

[0046] Data analysis: Cell proliferation rate (%) = [(OD sample group - OD blank group) / (OD solvent control group - OD blank group)] × 100% result: 1. Recombinant humanized type XVII collagen-1 ( Figure 4 ): The most promising proliferative active ingredient Trend analysis: Cell viability showed a typical dose-dependent increase within the concentration range of 0.097 ~ 12.5 μg / mL. The highest viability of 120.1% was reached at 12.5 μg / mL, representing an improvement of approximately 20% compared to the control.

[0047] Conclusion: This collagen has a strong ability to promote cell proliferation at low concentrations, making it suitable as an active ingredient to promote wound healing or skin regeneration.

[0048] 2. Recombinant humanized type XVII collagen-2 ( Figure 5 Moderate proliferative effect Trend analysis: Within the range of 0.78 ~ 25 μg / mL, the activity steadily increased from 103.9% to 113.8% (peak at 25 μg / mL).

[0049] Conclusion: This collagen has a moderate proliferative effect, a wide effective concentration window, and good safety.

[0050] (2) Recombinant humanized type XVII collagen promotes HaCaT cell migration (scratch assay) (cell function level - barrier repair efficacy) Cell seeding: HaCaT cells were seeded at a high density (approximately 5 × 10⁻⁶). 5 Cells / wells were seeded in 6-well plates and cultured to 100% confluence after 24 hours.

[0051] Cell scratching: Using the tip of a 200 μL sterile pipette, perpendicular to the pre-drawn horizontal line on the back of the well plate, forcefully and at a constant speed, draw several straight lines. Gently wash three times with PBS to remove floating cells.

[0052] Photography: Replace the medium with serum-free or low-serum culture medium containing different concentrations of samples, including COL17-1 (12.5 μg / mL), COL17-2 (12.5 μg / mL), and a positive control (epidermal growth factor EGF, 10 μg / mL). Take photos at the same location under an inverted microscope at 0 hours and 24 hours (or 36 hours) for recording.

[0053] Image analysis and data processing: Use software such as ImageJ to measure the width or area of ​​the scratch at each time point.

[0054] Calculate the scratch healing rate: Scratch healing rate (migration rate) (%) = [(0h scratch area - 24h scratch area) / 0h scratch area] × 100% Results: Among the three collagen samples, recombinant humanized type XVII collagen-2 exhibited the most significant migration-promoting activity, with a migration rate of 19.0%, which was statistically significant compared with the blank control (p < 0.05), and the migration promotion rate was approximately 43.9%. The migration rate of the recombinant humanized type XVII collagen-1 treatment group was 15.2%, slightly higher than the blank control (+15.2%), but not statistically significant (p > 0.05). These results indicate that recombinant humanized type XVII collagen-2 has biological activity that promotes HaCat cell migration, suggesting its potential application value in promoting skin wound healing. Recombinant humanized type XVII collagen-1 did not show a significant migration-promoting effect under the experimental conditions.

[0055] In summary, recombinant humanized type XVII collagen-2 possesses both moderate proliferative activity (proliferation rate +13.8%) and significant migration-promoting activity (migration rate +43.9%), making it the most comprehensive candidate for promoting wound healing.

[0056] Recombinant humanized type XVII collagen-1: It has the strongest proliferative effect (+20.1%), but its ability to promote migration is limited, suggesting that its mechanism of action may focus on stimulating cell division and proliferation rather than inducing directional cell movement.

[0057] Example 6: Detection of antioxidant activity of recombinant humanized type XVII collagen In vitro antioxidant experiment: Prepare 0.1 mM DPPH solution: Prepare twice, each time take 0.002 g DPPH and dissolve in 50 mL ethanol, store in the dark.

[0058] Sample solutions of specific concentrations were prepared at dilution ratios of 1:64, 1:32, 1:16, 1:8, 1:4, and 1:2. Three groups of 96-well plates were prepared, with three replicates in each group. The amount of sample added per well and the distribution across the 96-well plates are as follows: Sample (sample group): 100 μL of sample solution + 100 μL of DPPH alcohol solution (3 wells per concentration); blank group: 100uL of sample solution + 100uL of anhydrous ethanol (3 wells per concentration); control group: 100 μL DPPH alcohol solution + 100 μL anhydrous ethanol (one control group can be used on one plate, 3 wells). Spotting (operate in the dark; after spotting, incubate at room temperature in the dark for 30 minutes, and measure absorbance OD517).

[0059] The formula for calculating clearance rate (inhibition rate) is as follows: Inhibition%=[1-(sample-blank) / control]×100% Results: Antioxidant activity assays showed that, among the two collagen samples, recombinant humanized type XVII collagen-2 exhibited the strongest free radical scavenging ability at the highest concentration (0.4 mg / mL), with an antioxidant rate of 66.08%, which was higher than the 58.55% of recombinant humanized type XVII collagen-1 (0.2 mg / mL). Further analysis using gradient dilution assays determined the half-maximal effective concentration (EC50). 50 It was discovered that the EC of recombinant humanized type XVII collagen-1 50 The concentration was 0.148 mg / mL, significantly lower than the 0.260 mg / mL of recombinant humanized type XVII collagen-2, indicating that recombinant humanized type XVII collagen-1 can exert antioxidant effects at lower concentrations and has higher antioxidant efficacy. Both exhibit concentration-dependent antioxidant activity, with stronger activity above 0.1 mg / mL and activity essentially disappearing below 0.025 mg / mL. In summary, recombinant humanized type XVII collagen-1 combines strong maximum concentration activity with superior efficacy (low EC50). 50 ), is the most promising antioxidant candidate; recombinant humanized type XVII collagen-2 has outstanding antioxidant activity at higher concentrations, but the effective concentration is relatively high.

Claims

1. A recombinant humanized collagen type XVII, characterized in that, The amino acid sequence of the collagen is shown in either (A) or (B) below: (A) The amino acid sequence as shown in SEQ ID NO. 1; (B) The amino acid sequence as shown in SEQ ID NO. 2; The collagen's antioxidant activity and / or barrier repair activity.

2. The recombinant humanized collagen type XVII according to claim 1, characterized in that, The collagen also includes a functional variant having ≥80% sequence homology with the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the functional variant retains the antioxidant activity and / or barrier repair activity of the collagen.

3. The recombinant humanized collagen type XVII according to claim 1 or 2, characterized in that, The amino acid sequence also includes a derived sequence obtained by substitution, deletion, insertion or addition of one or more amino acid residues based on the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the derived sequence has collagen biological activity.

4. The recombinant humanized collagen type XVII according to claim 1 or 2 or 3, characterized in that, The amino acid sequence of the collagen consists of repeating units as shown in (A)n and / or (B)n, or contains the repeating units; wherein n is an integer ≥1, preferably an integer from 1 to 10, and more preferably an integer from 1 to 5.

5. A nucleic acid molecule encoding the recombinant humanized type XVII collagen as described in any one of claims 1-4.

6. A recombinant expression vector containing the nucleic acid molecule of claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or the recombinant expression vector of claim 6, characterized in that, The host cell is Pichia pastoris cell, Escherichia coli, or other host cells of recombinant protein expression systems.

8. A method for preparing recombinant humanized type XVII collagen, characterized in that, The method includes the following steps: (1) Construct an expression vector containing a nucleic acid molecule encoding the recombinant humanized type XVII collagen as described in any one of claims 1-4; (2) Transform the expression vector described in step (1) into host cells of Pichia pastoris, Escherichia coli, or other recombinant protein expression systems; (3) Inducing the expression of recombinant humanized type XVII collagen in the host cells described in step (2); (4) Collect and purify the expressed recombinant humanized type XVII collagen.

9. An application of a recombinant humanized type XVII collagen, characterized in that, The recombinant humanized type XVII collagen is the recombinant humanized type XVII collagen according to any one of claims 1-4, or the recombinant humanized type XVII collagen prepared by the method of claim 8, and the application is in the preparation of products with antioxidant function and / or skin barrier repair function.

10. The application of the recombinant humanized type XVII collagen according to claim 9, characterized in that, The products mentioned are skin care products, cosmetics, medical dressings, or pharmaceutical preparations.

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