Anti-photoaging recombinant human collagen type xvii and preparation method and application thereof

Abstract

The present application relates to the technical field of synthetic biology, in particular to an anti-photoaging recombinant human type XVII collagen as well as a preparation method and application thereof.The anti-photoaging recombinant human type XVII collagen in the present application is obtained by repeating a basic repeating unit n times, the amino acid sequence of the basic repeating unit is shown as SEQ ID NO.1, and n is a positive integer greater than or equal to 1.Further, the value range of the repeating number n of the basic repeating unit is 16<=n<=20.The anti-photoaging recombinant human type XVII collagen fragment synthesized in the present application has good anti-aging, anti-photoaging and anti-inflammatory effects, thereby enriching the type selection of anti-photoaging recombinant collagen.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, specifically to an anti-photoaging recombinant human type XVII collagen, its preparation method, and its applications. Background Technology

[0002] With the continuous deterioration of environmental problems and the gradual thinning of the ozone layer, the issue of ultraviolet radiation has become increasingly urgent, and skin aging related to ultraviolet damage has become a hot topic of concern. Solar ultraviolet radiation is the most important trigger for exogenous aging. Ultraviolet radiation induces oxidative stress, leading to changes in skin structure and is the core initiating factor in photoaging. When skin cells are damaged by oxidative stress, DNA damage, and mitochondrial damage caused by ultraviolet radiation, excessive collagen aging and a reduction in elastic fibers occur, resulting in photoaging of the skin.

[0003] Recombinant collagen is a type of collagen synthesized using bioengineering technology. It is highly similar to the body's own collagen and boasts advantages such as high safety and good biocompatibility. Recombinant collagen can repair UV damage by promoting skin cell proliferation and migration. However, for anti-photoaging effects, currently developed recombinant collagens are mainly based on type I and type III collagen. Therefore, developing more types of anti-photoaging recombinant collagen is of great significance. Summary of the Invention

[0004] The technical problem solved by this invention is to enrich the selection of recombinant collagen for anti-photoaging.

[0005] To address the aforementioned problems, as a first aspect, the present invention provides an anti-photoaging recombinant human type XVII collagen, wherein the anti-photoaging recombinant human type XVII collagen is obtained by repeating a basic repeating unit n times, and the amino acid sequence of the basic repeating unit is shown in SEQ ID NO.1, where n is a positive integer greater than or equal to 1.

[0006] Optionally, the number of repetitions n of the basic repeating unit is in the range of 16 ≤ n ≤ 20.

[0007] As a second aspect, the present invention provides a nucleic acid molecule that encodes the anti-photoaging recombinant human type XVII collagen as described above.

[0008] As a third aspect, the present invention provides an expression vector comprising the nucleic acid molecules described above.

[0009] Optionally, the expression vector is pPICZaA.

[0010] As a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecules described above, or comprising the expression vector described above, or expressing the anti-photoaging recombinant human type XVII collagen described above.

[0011] Optionally, the host cell is Escherichia coli or Pichia pastoris.

[0012] As a fifth aspect, the present invention provides a method for preparing recombinant human type XVII collagen for anti-photoaging, comprising: The anti-photoaging recombinant human type XVII collagen was expressed in host cells as described above to obtain cell cultures; The cell culture was isolated and purified to obtain the anti-photoaging recombinant human type XVII collagen.

[0013] As a sixth aspect, the present invention provides a product comprising the anti-photoaging recombinant human type XVII collagen as described above, or comprising anti-photoaging recombinant human type XVII collagen prepared by the preparation method described above.

[0014] As a seventh aspect, the present invention provides the application of the anti-photoaging recombinant human type XVII collagen as described above in the fields of pharmaceuticals, cosmetics or medical devices.

[0015] The advantages of this invention compared to related technologies include: The recombinant human type XVII collagen fragment for anti-photoaging in this invention exhibits excellent anti-aging, anti-photoaging, and anti-inflammatory effects, which is of great significance in filling the gap in the field of recombinant type XVII collagen for anti-photoaging. The recombinant human type XVII collagen for anti-photoaging in this invention can be fermented under normal conditions using a commercially available expression host, with high secretion yield, and does not produce adverse immune responses in humans. It demonstrates excellent anti-photoaging efficacy in both in vivo and in vitro experiments. Attached Figure Description

[0016] Figure 1 This is an image showing the SDS-PAGE electrophoresis results of the anti-photoaging recombinant human type XVII collagen in Example 1 of the present invention.

[0017] Figure 2a , Figure 2b This is an observation image of the skin aging status of animals in each group at the end of the comparative test of anti-photoaging effect in Example 4 of the present invention.

[0018] Figure 3a , Figure 3b HE-stained sections of animal skin tissue from each group at the final stage of the comparative test of anti-photoaging effects in Example 4 of this invention.

[0019] Figure 4a , Figure 4b These are MASSON stained sections of animal skin tissue from each group at the final stage of the comparative experiment on anti-photoaging effects in Example 4 of this invention.

[0020] Figure 5 This is a comparison chart of MDA content in the supernatant of HaCat cells in each group during the oxidative inhibition evaluation experiment in Example 5 of this invention; Figure 6 This is a comparison of IL-6 mRNA expression levels in the supernatant of HaCat cells in each group during the oxidative inhibition evaluation experiment in Example 5 of this invention. Figure 7 This is a comparison of IL-1β mRNA expression levels in the supernatant of HaCat cells in each group during the oxidative inhibition evaluation experiment in Example 5 of this invention. Detailed Implementation

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0022] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used in the description of this application is for the purpose of describing particular embodiments only and is not intended to limit this application. The term "comprising" and its variations as used herein are open-ended, meaning "including but not limited to"; the term "based on" means "at least partially based on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one additional embodiment"; the term "some embodiments" means "at least some embodiments"; and the term "optionally" means "optional embodiments". Definitions of other terms will be given in the description below. It should be noted that the concepts of "first," "second," etc., mentioned in this invention are used to distinguish different objects, not to describe a specific order or hierarchy. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0023] To enrich the selection of recombinant collagen for anti-photoaging, this invention provides a recombinant human type XVII collagen for anti-photoaging, which is obtained by repeating a basic repeating unit n times, where n is a positive integer greater than or equal to 1. The amino acid sequence of the basic repeating unit is shown in SEQ ID NO.1: SEQ ID NO. 1: GPRGPPGPPGASGDG.

[0024] The inventors discovered that the aforementioned recombinant human type XVII collagen fragment for anti-photoaging possesses excellent anti-aging, anti-photoaging, and anti-inflammatory effects. The recombinant human type XVII collagen of this invention can be fermented under conventional conditions using a commercially available expression host, exhibiting high secretion yield, and demonstrates excellent anti-photoaging and anti-inflammatory effects in both in vivo and in vitro experiments.

[0025] In some optional embodiments, the number of repetitions n of the basic repeating unit is in the range of 16 ≤ n ≤ 20.

[0026] It is understood that the basic repeating unit of recombinant collagen refers to the periodically repeating amino acid sequence in its peptide chain, which directly determines the triple helix structure and biological function of the molecule. For recombinant humanized collagen, the precise design of the repeating unit is crucial to the recombinant technology. By rationally designing the number of repetitions of the basic repeating unit, the structural and functional requirements of recombinant collagen, production process optimization, and comprehensive consideration of product application scenarios can be met. The inventors have discovered that the anti-photoaging recombinant human type XVII collagen of this invention, within the aforementioned number of repetitions, can ensure biological activity while reducing production complexity, achieving a balance between expression efficiency and molecular weight, and improving the feasibility of genetic engineering.

[0027] As an example, the number of repetitions n of the basic repeating unit can be 16, in which case the amino acid sequence of the anti-photoaging recombinant human type XVII collagen is shown in SEQ ID NO.2: SEQ ID NO.2: GPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDG GPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDG.

[0028] In another embodiment, the number of repetitions n of the basic repeating unit can be 20, in which case the amino acid sequence of the anti-photoaging recombinant human type XVII collagen is as shown in SEQ ID NO.3: SEQ ID NO.3: GPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDG GPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDGGPRGPPGPPGASGDG.

[0029] Another embodiment of the present invention provides a nucleic acid molecule that encodes the anti-photoaging recombinant human type XVII collagen as described above.

[0030] Nucleic acid molecules, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), provide the most fundamental coding information. It should be understood that, based on the amino acid sequence of the aforementioned anti-photoaging recombinant human type XVII collagen, all nucleic acid molecules capable of encoding the above-mentioned amino acid sequence are within the scope of protection of this invention. Furthermore, based on the codon preferences of host cells, the relevant gene sequences of nucleic acid molecules can be modified and optimized, thereby enabling efficient translation by the host without altering the amino acid sequence, according to the codon preferences of different host cells.

[0031] Another embodiment of the present invention provides an expression vector comprising the nucleic acid molecule described above. The expression vector is preferably pPICZaA.

[0032] An expression vector is an artificially constructed DNA molecule that can self-replicate in a host cell and drive the efficient transcription and translation of the target gene it carries within the cell, ultimately producing the corresponding protein. In the preparation of recombinant collagen, the optimized anti-photoaging recombinant human type XVII collagen nucleic acid molecule is inserted into the multiple cloning site of the expression vector to construct the recombinant expression vector. This recombinant expression vector is then transferred into host cells to generate the protein.

[0033] Another embodiment of the present invention provides a host cell comprising the nucleic acid molecules described above, or comprising the expression vector described above, or expressing the anti-photoaging recombinant human type XVII collagen described above.

[0034] It should be understood that, in this embodiment, the host cell refers to a living cell into which anti-photoaging recombinant human type XVII collagen nucleic acid molecules have been introduced and which utilize their endogenous transcription and translation mechanisms to produce the corresponding anti-photoaging recombinant human type XVII collagen. The host cell provides necessary components such as ribosomes, transfer RNA, enzymes, and energy during protein encoding, converting the genetic information of the exogenous gene into protein. It should be noted that the aforementioned host cell can be a prokaryotic or 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. Preferably, the host cell in this embodiment is *Escherichia coli* or *Pichia pastoris*.

[0035] Another embodiment of the present invention provides a method for preparing anti-photoaging recombinant human type XVII collagen, comprising: expressing anti-photoaging recombinant human type XVII collagen in host cells as described above, obtaining cell culture, separating and purifying the cell culture to obtain anti-photoaging recombinant human type XVII collagen.

[0036] The present invention does not impose any restrictions on the expression method, which can be selected and adjusted as needed. Specifically, the expression can be one or a combination of two of the following: compositional expression and induced expression. The inducing agent for induced expression can be IPTG, β-galactoside, methanol, ethanol, etc. Salting out, chromatographic chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, acid-base precipitation, and membrane separation are used, preferably a combination of chromatographic chromatography and membrane separation, and more preferably a combination of ion exchange chromatography and membrane separation.

[0037] Another embodiment of the present invention provides a product comprising, as described above, recombinant human type XVII collagen for anti-photoaging, or recombinant human type XVII collagen for anti-photoaging prepared by the preparation method described above. Exemplarily, the product may be a collagen composition or skincare product prepared by mixing the above-mentioned recombinant human type XVII collagen with components such as vitamin E, antioxidants, glycerin, ginseng extract, and astragalus extract.

[0038] Furthermore, some embodiments of the present invention also provide the application of the above-mentioned anti-photoaging recombinant human type XVII collagen in the fields of pharmaceuticals, cosmetics, or medical devices. Specifically, the above-mentioned pharmaceuticals, cosmetics, or medical devices are used for anti-photoaging effects.

[0039] The present invention will be described in detail below through specific embodiments and comparative examples: Example 1 (1) Construction of pPICZaA-HLC expression strain The amino acid sequence, as shown in SEQ ID No. 2, which is repeated 16 times based on the codon preference of Pichia pastoris, was synthesized through whole-genome synthesis and directly linked to the pPICZaA plasmid. Subsequently, the pPICZaA plasmid was transformed into DE3 competent cells by heat shock, plated on resistant plates, and single colonies picked from the plates were the yeast expression strains.

[0040] (2) Expression of recombinant human type XVII collagen for anti-photoaging First, the yeast expression strain was inoculated into 50 ml of YPD liquid medium and cultured at 30℃. When the OD600 reached 4-8, it could be used as a fermentation seed liquid.

[0041] The fermenter parameters were set as follows: temperature 30℃, rotation speed 200-700 rpm for 2 days, aeration rate 4 L / min (automatically adjusted by the fermenter), dissolved oxygen maintained above 30%, and pH adjusted to 4.5 using ammonia. The above-mentioned seed culture was then transferred to a sterile fermenter to begin fermentation. Once the carbon source was depleted, feed culture medium was added. Fermentation was terminated when the wet weight of the cells reached 220 g / L.

[0042] Collect the fermentation broth and prepare a 10% (g wet cells / mL PBS) bacterial suspension with PBS buffer at pH=6.0. Homogenize the suspension at 800 bar for 3 min, centrifuge at 9000 rpm for 10 min, and collect the supernatant, which is the crude protein solution.

[0043] (3) Isolation and purification of recombinant human type XVII collagen for anti-photoaging Add 60% NaCl to the crude protein solution collected by centrifugation, stir to dissolve, let stand at room temperature for 2 hours, centrifuge at 9000 rpm for 10 minutes, and collect the supernatant. Concentrate and desalt the collected supernatant through a 30KD ultrafiltration membrane, adjust the pH to 6.5 with phosphoric acid, load the solution onto a cation exchange resin, and elute with 0.5M NaCl to obtain the target protein solution.

[0044] The collected target protein solution was desalted by a 30KD ultrafiltration membrane, then pre-frozen at -20℃ for 4 hours, and then transferred to a vacuum freeze dryer for lyophilization. The lyophilized protein was collected after 48 hours.

[0045] The SDS-PAGE electrophoresis results of the anti-photoaging recombinant human type XVII collagen in this embodiment are as follows: Figure 1 As shown. Figure 1 The rightmost image shows a clear molecular weight standard gradient band. Using a protein molecular weight standard as a reference, a clear band is visible near 30 kDa in the sample lane. By comparing the molecular weight, it can be determined that the expressed protein is the target amino acid sequence.

[0046] Example 2 (1) Construction of pPICZaA-HLC expression strain The amino acid sequence, as shown in SEQ ID No. 3, which is repeated 20 times based on the codon preference of Pichia pastoris, was synthesized by whole-genome synthesis and directly linked to the pPICZaA plasmid. Subsequently, the pPICZaA plasmid was transformed into DE3 competent cells by heat shock, plated on resistant plates, and single colonies picked from the plates were the yeast expression strains.

[0047] (2) Expression of recombinant human type XVII collagen for anti-photoaging First, the yeast expression strain was inoculated into 50 ml of YPD liquid medium and cultured at 32℃. When the OD600 reached 6 to 8, it could be used as a fermentation seed liquid.

[0048] The fermenter parameters were set as follows: temperature 30℃, rotation speed 250-750 rpm for 2 days, aeration rate 4 L / min (automatically adjusted by the fermenter), dissolved oxygen maintained above 30%, and pH adjusted to 4.5 using ammonia. The above-mentioned seed culture was then transferred to a sterile fermenter to begin fermentation. Once the carbon source was depleted, feed culture medium was added. Fermentation was terminated when the cell wet weight reached 220 g / L.

[0049] Collect the fermentation broth and prepare a 10% (g wet cells / mL PBS) bacterial suspension with PBS buffer at pH=6.0. Homogenize the suspension at 800 bar for 3 min, centrifuge at 9000 rpm for 10 min, and collect the supernatant, which is the crude protein solution.

[0050] (3) Isolation and purification of recombinant human type XVII collagen for anti-photoaging The separation and purification process of the crude protein solution is the same as in Example 1.

[0051] Example 3 This embodiment provides an anti-photoaging recombinant human type XVII collagen composition: Weigh 25% of the anti-photoaging recombinant human type XVII collagen prepared in Example 1, 6% vitamin E, 2% antioxidant, 5% fibroblasts, 3% glycerol, 3% carbomer and 3% triethanolamine by mass percentage and put them into a reaction vessel for mixing. At the same time, deionized water is added in small amounts several times. The temperature of the reaction vessel is heated to 40°C. After the raw materials and deionized water are completely mixed, stock solution A is obtained. The heating of the reaction vessel is stopped and stock solution A is allowed to cool naturally.

[0052] Weigh out 4% sodium chloride dilution, 8% potassium dihydrogen phosphate, 5% dimethyl hydrogen phosphate buffer, 8% sodium hyaluronate, 5% ginseng extract, and 5% astragalus extract by mass percentage and mix them in a reaction vessel. Add deionized water in small amounts several times. Heat the reaction vessel to 55°C. After the raw materials and deionized water are completely mixed, the stock solution B is obtained. Stop heating the reaction vessel and allow the stock solution B to cool naturally.

[0053] Stock solutions A and B were poured into an emulsifying tank at a volume ratio of 2:1 under sterile conditions at 32°C and stirred until homogeneous. Then, an emulsifier was added to obtain a mixture of stock solutions A and B. After the mixture was allowed to stand for 1 hour, a pH adjuster was added to adjust the pH value to 7.3. The mixture was stirred for 20 minutes and then allowed to cool naturally to obtain an anti-photoaging recombinant human type XVII collagen composition.

[0054] Example 4 A comparative experiment was conducted using the recombinant collagen of type XVII or its composition from Examples 1 to 3 with commercially available anti-photoaging products to assess their anti-photoaging effects. Select 100 young adult mice (100 ICR mice, SPF grade, male, weight: 18-22g, age: about 6 weeks), and the weight difference of all animals used in the experiment should be within ±20% of the average weight of each sex.

[0055] The experiment was conducted using the facility's barrier system, with a 12h / 12h day / night cycle, ≥15 air changes per hour, a temperature range of 20℃ to 26℃, a pressure gradient of ≥10Pa, and a relative humidity range of 40% to 70%. Monitoring and recording were performed daily. Mice arrived at the laboratory and underwent quarantine before being placed in a 3-day acclimatization room in polypropylene (PP) cages with sturdy bottoms and bedding. The bedding material (wood shavings) was changed weekly. Water was provided freely to the animals. Based on sex and weight, the mice were randomly divided into 9 groups of 10 mice each using a random grouping and recording method. The groups were: blank control group, ultraviolet (UVB) treatment group, anti-photoaging recombinant human type XVII collagen and its composition treatment group in Examples 1 to 3, and comparative examples 1 to 4 drug treatment group.

[0056] Modeling: Before modeling, the skin condition of the animals needed to be confirmed, with most animals (90%) showing pinkish-white skin. The day before modeling, all animals underwent skin preparation. Except for the blank control group, mice were anesthetized with isoflurane, and the hair in the middle of the spinal region on the back of the mice, covering an area of ​​approximately 1.5 cm * 1.5 cm, was shaved off. Residual hair was removed with depilatory cream. The specific treatment methods for each group of mice 24 hours after hair removal are shown in Table 1. Table 1. Treatment methods for mice in each group in the comparison test of anti-photoaging effects.

[0057] Apply the above medication once a day for 28 consecutive days. The topical medications for the above experimental groups were prepared using fresh basal culture medium. Example 1 treatment group: The aqueous solution of anti-photoaging recombinant human type XVII collagen prepared in Example 1, with a protein concentration of 2 mg / ml; Example 2 treatment group: The aqueous solution of anti-photoaging recombinant human type XVII collagen prepared in Example 2, with a protein concentration of 2 mg / ml; Example 3 treatment group: The anti-photoaging recombinant human type XVII collagen composition prepared in Example 3, with a composition concentration of 2 mg / ml; Comparative Example 1 Treatment Group: A mixed aqueous solution of VE (Shanghai Hewu Biotechnology Co., Ltd.) and VC (Guangzhou Suxiuchenshi Biotechnology Co., Ltd.) with a concentration of 2 mg / ml.

[0058] Comparative Example 2: Super A Peptide Anti-aging Revitalizing Essence (Huameike).

[0059] Comparative Example 3 Treatment Group: AROMATHERBS Recombinant Collagen Single-use Essence (AROMATHERBS).

[0060] Comparative Example 4: ZO SKIN HEALTH Antioxidant Daily Essence UV Defense Repair Essence (ZO SKINHEALTH). Mice in each group were observed daily after treatment, and photographed weekly after the skin began to show signs of aging and wrinkling. Three areas of aged skin were randomly selected from each group of mice, and the skin barrier water loss ratio (TWEL) was measured and statistically analyzed. The results are shown in Table 2. Table 2. Statistical results of skin barrier water loss rate (TWEL) values ​​in each group of mice.

[0061] As shown in Table 2, the skin water loss rate of mice in the Comparative Examples 1 to 4 treatment groups was slightly reduced compared to the UVB treatment group, while the skin water loss rate of mice in the Examples 1 to 3 treatment groups was significantly reduced compared to the UVB treatment group. The reduction in water loss rate was most significant in the Example 1 treatment group.

[0062] Animals were necropsy performed at the end of the drug administration period, and skin samples were collected. The skin aging status of each group of animals is shown in the following figures. Figure 2a , Figure 2b As shown. Subsequently, HE staining and MASSON staining were performed on the case sections, and the slides were reviewed. HE-stained sections of skin tissue from each group of animals are shown below. Figure 3a , Figure 3bAs shown, the MASSON-stained sections of skin tissue from each group of animals are as follows. Figure 4a , Figure 4b As shown.

[0063] from Figure 2a , Figure 2b It can be seen that the drugs used in the treatment groups of Comparative Examples 1 to 4 all have some photoaging repair effects, and the aging wrinkles and verification reactions at the animal skin preparation sites are repaired compared to the ultraviolet (UVB) treatment group. Furthermore, the type XVII collagen in the treatment groups of Examples 1 to 3 of this invention also has anti-wrinkle and anti-photoaging repair effects, and the type XVII collagen in the treatment group of Example 1 has better anti-wrinkle and anti-photoaging repair effects than the samples in Comparative Examples 1-4, completely repairing skin wrinkles.

[0064] from Figure 3a , Figure 3b It can be seen that in the HE-stained sections of mouse skin tissue from the ultraviolet-treated group, the epidermal layer of the skin tissue showed a thickening phenomenon. Similarly, the drugs used in the treatment groups of Comparative Examples 1 to 4 all had partial photoaging repair effects, and the thickness of the mouse epidermal layer was improved and thinned. The thickness of the mouse epidermal layer in the treatment groups of Examples 1 to 3 of this invention also decreased, and the epidermal layer of the mouse skin tissue in the treatment group of Example 1 was the thinnest, which also indicates that the type XVII collagen in Example 1 has better and higher anti-wrinkle and anti-photoaging repair effects.

[0065] from Figure 4a , Figure 4b It can be seen that the blue color of the MASSON-stained sections of mouse skin tissue in the treatment groups of Examples 1 to 3 is deeper than that of Comparative Examples 1-4 and the ultraviolet-treated group, indicating richer and denser collagen. Among them, the MASSON-stained sections in the treatment group of Example 1 are the richest and densest. This further illustrates that the type XVII collagen in Example 1 has better and higher anti-wrinkle and anti-photoaging repair effects.

[0066] Example 5 An evaluation experiment was conducted on the inhibitory effects of recombinant collagen of type XVII or a combination thereof from Examples 1 to 3, together with commercially available anti-photoaging products, on the oxidation inhibition of MDA, IL-6, and IL-1β in human epidermal keratinocytes (HACAT).

[0067] Inoculation: 1 mL of HACAT (human epidermal keratinocyte immortalized cells) cells were inoculated into each well of a 24-well plate (8 × 10⁴ cells / well) and incubated at 37°C and 5% CO₂ for 24 h. Modeling: After incubation, the culture medium was removed, and the cells were washed once or twice with PBS. The cells were then divided into 9 groups: a blank control group, an ultraviolet (UVB) treatment group, an anti-photoaging recombinant human type XVII collagen and its composition treatment group (Examples 1 to 3), and a drug treatment group (Comparative Examples 1 to 4). Except for the blank control group, each treatment group was given 200 μM testosterone in 1 mL of serum-free medium. After 1 hour, the medium was replaced with serum-containing medium, and after 3 hours of recovery, the medium was replaced with normal medium. Simultaneously, the blank control group was replaced with normal medium. Drug administration: After modeling, the blank control group was removed, and 1 mL of fresh basal culture medium was added to each well. Then, each treatment group was subjected to UVB and drug administration according to the drug administration method in Example 4 above, and incubated at 37°C and 5% CO2 for 24 h.

[0068] Cell supernatant was extracted, and the MDA content of each treatment group was detected using an ELISA kit. Subsequently, cells from each treatment group were digested, and cellular mRNA was extracted for Q-PCR, IL-6, and IL-1β detection. The results are shown below. Figures 5 to 7 As shown.

[0069] The samples described above were tested using the same method described above, and a blank control was set up. No antioxidant products were added to the blank control.

[0070] from Figure 5 It can be seen that the MDA in the samples of the comparative examples 1 to 4 was slightly reduced, indicating that the antioxidants used in them all had some MDA inhibition. The samples of the examples 1 to 3 had better MDA generation inhibition compared to the comparative examples 1 to 4, and the MDA in the example 1 treatment group was significantly reduced. This shows that the type XVII collagen of the present invention has a better MDA inhibition effect.

[0071] from Figure 6 , 7 It can be seen that the mRNA inflammatory factors of IL-6 and IL-1β were slightly reduced in the samples of Comparative Examples 1 to 4, indicating that the corresponding antioxidants used could partially inhibit the production of IL-6 and IL-1β mRNA inflammatory factors. The samples of Examples 1 to 3 showed better inhibition of IL-6 and IL-1β mRNA production compared to Comparative Examples 1 to 4, and the mRNA inflammatory factors of IL-6 and IL-1β were significantly reduced in the Example 1 treatment group. This indicates that the type XVII collagen of Example 1 of this invention has a better inhibitory effect on mRNA inflammatory factors.

[0072] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A recombinant human type XVII collagen for anti-photoaging, characterized in that, The anti-photoaging recombinant human type XVII collagen is obtained by repeating a basic repeating unit n times. The amino acid sequence of the basic repeating unit is shown in SEQ ID NO.1, where n is a positive integer greater than or equal to 1.

2. The anti-photoaging recombinant human type XVII collagen according to claim 1, characterized in that, The number of repetitions n of the basic repeating unit is in the range of 16≤n≤20.

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

4. An expression carrier, characterized in that, The expression vector includes the nucleic acid molecule as described in claim 3.

5. The expression vector according to claim 4, characterized in that, The expression vector is pPICZaA.

6. A host cell, characterized in that, The host cell includes the nucleic acid molecule as described in claim 3, or the expression vector as described in claim 4 or 5, or expresses the anti-photoaging recombinant human type XVII collagen as described in claim 1 or 2.

7. The host cell according to claim 6, characterized in that, The host cell is either Escherichia coli or Pichia pastoris.

8. A method for preparing recombinant human type XVII collagen for anti-photoaging, characterized in that, include: The host cells described in claim 6 or 7 are used to express the anti-photoaging recombinant human type XVII collagen as described in claim 1 or 2 to obtain cell cultures; The cell culture was isolated and purified to obtain the anti-photoaging recombinant human type XVII collagen.

9. A product characterized in that, The product includes the anti-photoaging recombinant human type XVII collagen as described in claim 1 or 2, or includes the anti-photoaging recombinant human type XVII collagen prepared by the preparation method described in claim 8.

10. The application of the anti-photoaging recombinant human type XVII collagen as described in claim 1 or 2 in the fields of pharmaceuticals, cosmetics or medical devices.

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