A novel recombinant collagen and its use

Abstract

The present invention relates to the field of genetic engineering, protein engineering technology and its application, and discloses a novel recombinant collagen and its application, including any one of the recombinants COL17-CO-1, COL17-CO-2, COL17-CO-3 or COL17-CO-4 of the specific functional region of human type XVII collagen and the 1232-1466 region of human type III collagen, wherein the amino acid sequences of the specific functional regions in the COL17-CO-1, COL17-CO-2, COL17-CO-3 and COL17-CO-4 are SEQ ID No.2, SEQ ID No.6, SEQ ID No.8 and SEQ ID No.10, respectively. The recombinant exhibits better ability to promote cell proliferation, promotes the expression of proline hydroxylase (HYP), improves the antioxidant capacity of skin cells, improves the expression of multiple collagens in skin cells, and better reduces the expression of collagen degradation-related bases MMP-3 and MMP-9.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, protein engineering technology and their application, and in particular to a novel recombinant collagen and its application. Background Art

[0002] Type ⅩⅦ collagen (COL17) is a transmembrane protein in the basal layer of the epidermis. It has the function of maintaining a tight connection between the epidermis and the dermis to promote epidermal replacement and stabilize the basement membrane. Studies have found that COL17 has the function of regulating hair loss, skin aging, and wound repair. Mutations or deficiencies will lead to skin atrophy, delayed wound healing, and hair loss. Type III collagen accounts for about 10-15% of the skin content and is mainly distributed in the superficial dermis. In human infancy, type III collagen is abundant, but its content decreases after adulthood. The fiber morphology of type III collagen is relatively fine and presents a sparse mesh. The lack of type III collagen will lead to a decrease in the firmness and elasticity of the skin.

[0003] At present, type XVII collagen and type III collagen have certain records of use and effects in the fields of skin beauty, biomedical materials, etc. However, the recombinant type III collagen currently used usually adopts the core triple helix structure part, which is considered to be the core area of ​​type III collagen in cell adhesion, cell support, etc., and its role in anti-inflammatory is very limited. In addition, type XVII collagen is often used as a whole, and the effects of both are limited during use. There are no records of related drug development for other parts of type III collagen and type XVII collagen, resulting in very limited research and development of the structure and function of type XVII collagen and type III collagen.

[0004] In view of this, the development of a new type of recombinant collagen and its use in the preparation of anti-inflammatory, antioxidant and anti-aging drugs will not only effectively make up for the shortcomings of existing technologies, but also further expand the use of recombinant collagen in food, cosmetics, medical beauty, health products, pharmaceuticals and medical devices, etc., which is of great significance to the development of new drugs. Summary of the invention

[0005] The present invention is intended to provide a novel recombinant collagen and its use, so as to solve the technical problem that the research and development of collagen structure and function in the prior art is very limited.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a new type of recombinant collagen, including any one of the recombinants COL17-CO-1, COL17-CO-2, COL17-CO-3 or COL17-CO-4 of the specific functional region of human type XVII collagen and the 1232-1466 region of human type III collagen, wherein the amino acid sequences of the specific functional regions in COL17-CO-1, COL17-CO-2, COL17-CO-3 and COL17-CO-4 are SEQ ID No.2, SEQ ID No.6, SEQ ID No.8 and SEQ ID No.10 respectively.

[0007] Preferably, as an improvement, the amino acid sequence of the region 1232 to 1466 of the human type III collagen is SEQ ID No. 3.

[0008] Preferably, as an improvement, a linker is further included between the specific functional region of human type XVII collagen and the region 1232-1466 of human type III collagen, the amino acid sequence of the linker is SEQ ID No.4, and the recombinant amino acid sequence is any one of SEQ ID No.1, SEQ ID No.5, SEQ ID No.7 or SEQ ID No.9.

[0009] Preferably, as an improvement, the present scheme also provides a composition comprising a combination of any one of the recombinants COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4 and seaweed oligosaccharides.

[0010] Preferably, as an improvement, the present embodiment further provides a nucleic acid molecule, comprising a nucleic acid molecule encoding any one of the above-mentioned recombinant collagens COL17-CO-1, COL17-CO-2, COL17-CO-3 or COL17-CO-4, and the nucleotide sequences of the nucleic acid molecules encoding the recombinant collagens COL17-CO-1, COL17-CO-2, COL17-CO-3 or COL17-CO-4 are SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, and SEQ ID No.14, respectively.

[0011] Preferably, as an improvement, the present solution also provides an expression cassette comprising the above-mentioned nucleic acid molecule.

[0012] Preferably, as an improvement, the present solution also provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0013] Preferably, as an improvement, the present invention provides an expression system, comprising a host cell, wherein the host cell comprises any one of the above-mentioned recombinant collagen, the above-mentioned nucleic acid molecule, the above-mentioned expression cassette or the above-mentioned expression vector.

[0014] Preferably, as an improvement, the present solution also provides a use of a novel recombinant collagen, including the use of any one of the above-mentioned recombinant collagen and composition in the preparation of anti-aging drugs.

[0015] Preferably, as an improvement, the present solution also provides a use of a novel recombinant collagen, including the use of any one of the above-mentioned recombinant collagen and composition in the preparation of anti-aging drugs.

[0016] Preferably, as an improvement, the present solution also provides a use of a novel recombinant collagen, including the use of any one of the above-mentioned recombinant collagen and composition in the preparation of anti-inflammatory drugs.

[0017] Preferably, as an improvement, the present solution also provides a use of a novel recombinant collagen, including the use of any one of the above-mentioned recombinant collagen and composition in the preparation of antioxidant drugs.

[0018] Preferably, as an improvement, the present solution also provides a use of a novel recombinant collagen, including use of any one of the above-mentioned recombinant collagen and composition in the preparation of a hair growth drug.

[0019] Preferably, as an improvement, the present scheme also provides a use of a novel recombinant collagen, including the use of any one of the above-mentioned recombinant collagen and the composition in the preparation of any one of cosmetic preparations, daily chemical preparations, medical cosmetology, medical devices or biopharmaceutical preparations.

[0020] The principles and advantages of this solution are:

[0021] 1. This scheme obtains recombinant collagen COL17-CO-1, COL17-CO-2, COL17-CO-3, and COL17-CO-4 by recombining specific functional regions of human type XVII collagen and fragments of type III collagen. They not only have the function of promoting cell proliferation, but also can significantly promote the expression of genes related to collagen synthesis, while reducing the expression of genes related to collagen degradation and increasing the content of various collagens.

[0022] 2. The recombinant collagen of this scheme is effective in resisting skin cell aging and inflammation, and its combination with seaweed oligosaccharides can also effectively resist oxidation. In addition, the recombinant collagen of this scheme can effectively reduce the apoptosis of hair follicle cells, thereby effectively promoting hair growth in hair follicle cells. Specifically, on the 7th day of culture, the average number of apoptotic cells in each hair follicle in the control group and the COL17-CO-2 test group was 16.4±0.58 and 7.2±0.41, respectively; on the 7th day of culture, the average hair growth length of the hair follicles in the control group and the test group was (2.10±0.03) mm and (2.453±0.03) mm, respectively, with significant differences, which fully demonstrates that the COL17-CO-2 test group has the best effect in promoting the average hair growth of hair follicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the molecular weight electrophoresis diagram of the recombinant collagen purified in Example 1 of the present invention.

[0024] Figure 2 This is a graph showing the result of the promoting effect of recombinant collagen on HDFn cell proliferation in Experimental Example 1 of the present invention.

[0025] Figure 3 This is a graph showing the effect of recombinant collagen on collagen synthesis and degradation genes in Experimental Example 1 of the present invention.

[0026] Figure 4 This is a graph showing the effect of the combination of recombinant collagen and algae oligosaccharides on cell viability in Experimental Example 1 of the present invention.

[0027] Figure 5 This is a graph showing the antioxidant effect of the composition of recombinant collagen and seaweed oligosaccharides in Experimental Example 1 of the present invention.

[0028] Figure 6 This is a graph showing the effect of recombinant collagen on the expression of inflammatory factors in Experimental Example 1 of the present invention.

[0029] Figure 7 This is a graph showing the effect of the recombinant collagen COL17-CO-2 on hair follicle cells in Experimental Example 1 of the present invention.

[0030] Figure 8 This is a graph showing the effect of the recombinant collagen COL17-CO-3 on animal skin aging in Experimental Example 1 of the present invention.

[0031] Fig. 9 This is a graph showing the results of the recombinant collagen COL17-CO-3 repairing skin collagen loss in Experimental Example 1 of the present invention.

[0032] Fig.10 This is a graph showing the effect of recombinant collagen COL17-CO-3 on the expression of aging-related factors in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0034] Example 1

[0035] This scheme provides a novel recombinant collagen, including any one of the recombinant COL17-CO-1, COL17-CO-2, COL17-CO-3 or COL17-CO-4 of the specific functional region of human type XVII collagen and the region 1232 to 1466 of human type III collagen. The nucleotide sequence and amino acid sequence mentioned in this scheme are shown in Table 1:

[0036] Table 1 Sequence content

[0037]

[0038] Experimental Example 1: Recombinant collagen

[0039] 1. Vector construction and protein expression

[0040] The gene codons were optimized based on the frequency of Pichia pastoris codon usage, and codons with low usage rates were eliminated. A NotI (GCGGCCGC) restriction site and a histidine tag (HHHHHH) were added to the 3' end. The optimized gene sequence was synthesized by Qingke Biotechnology Co., Ltd. and then seamlessly connected to the NotI site of the pPIC9K vector for sequencing identification.

[0041] The present invention uses the Pichia pastoris SMD1168 strain as the expression host strain. The culture conditions used are as follows:

[0042] YPD complete medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose (solid medium contains 2% agar); MD medium (selective medium): prepare 100 mL, add 2 g agarose to 80 mL water, sterilize at 121°C for 20 minutes, wait until the temperature drops to 60°C, then add 10 mL 10×YNB, 10 mL 10×glucose, and 0.2 mL 500×biotin on a clean bench;

[0043] BMGY medium (yeast growth medium): completely dissolve 10g yeast extract, 20g peptone, 3g K2HPO4, 11.8g KH2PO4, and adjust the volume to 890mL. Autoclave for 20min, cool to 60℃, then add 100mL 10×YNB, 1mL 500×biotin, and 10mL glycerol on a clean bench;

[0044] BMMY medium: completely dissolve 10g yeast extract, 20g peptone, 3g K2HPO4, 11.8g KH2PO4, and adjust the volume to 895mL. Sterilize by steam autoclave at 121℃ for 20min, cool to 60℃, and add 100mL 10×YNB, 1mL 500×biotin, and 5mL methanol on a clean bench.

[0045] Linearization of expression vectors pPIC9K-His-COL17-CO-4, pPIC9K-His-COL17-CO-4, pPIC9K-His-COL17-CO-4, pPIC9K-His-COL17-CO-4, pPIC9K-His-COLF1: digested with restriction endonuclease SalI at 37°C overnight, the reaction system is as follows: plasmid 10 μg, 10×H buffer 5 μL, SalI 50U sterile water to 200 μL. Cut at 16°C overnight, add loading buffer, detect by 0.7% agarose gel electrophoresis, and recover the corresponding bands with PCR product purification kit (Omega).

[0046] Preparation of competent cells of Pichia pastoris SMD1168: Pick a single colony of yeast SMD1168, inoculate it into a test tube containing 5 mL of YPD liquid medium, and culture it with shaking at 30°C and 220 rpm overnight; take 50 μL of the overnight culture and inoculate it into a 500 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, and culture it with shaking at 30°C and 220 rpm overnight until the OD600 value reaches 1.1-1.3; centrifuge the culture at 4°C, 1500×g for 5 min, and resuspend the bacteria with 50 mL of ice-cold sterile double distilled water; centrifuge, resuspend the bacteria with 25 mL of ice-cold sterile double distilled water; centrifuge, resuspend the bacteria with 20 mL of ice-cold 1M sorbitol solution; centrifuge, resuspend the bacteria with 0.3 mL of ice-cold 1M sorbitol solution, and the final volume is about 0.5 mL; aliquot into 80 μL portions and store at -70°C for later use.

[0047] Electrotransformation of Pichia pastoris: Place a 0.2 cm electroporation cuvette in ice for 10 min; add about 10 μL of linearized plasmid to freshly prepared Pichia pastoris competent cells, mix gently, transfer to a 0.2 cm ice-precooled electroporation cuvette, and continue to precool on ice for 5 min; electroporation, voltage 1.5 kV; capacitance 25 μF; resistance 200 Ω; electroporation time 5-10 mSec; after electroporation, quickly add 1 mL of ice-precooled 1 M sorbitol solution, gently blow to mix, transfer to a 1.5 mL centrifuge tube, and incubate at 30°C for 1 h; spread the bacterial suspension on the MD plate, spread one plate for every 100 μL-200 μL, let stand at room temperature for 10 min, and invert and culture at 30°C for 2-5 days until a single colony appears.

[0048] Screening of copy-insertion recombinants: Add 2 mL of sterile double-distilled water to the surface of the MD plate where transformants grow, then gently scrape the His+ transformants on the surface of the plate with a sterile triangular applicator and transfer them to a 50 mL centrifuge tube; add 20 mL of sterile double-distilled water to dilute, mix well and measure its OD600 value (1OD = 5 × 107 cells / mL); take 108 cells and spread them on a YPD plate containing 0.5 mg / mL G418, invert, and culture at 30°C for 3-4 days; add 200 μL of YPD liquid culture medium to each well of a sterile 96-well plate; use a sterile toothpick to inoculate the transformants obtained on the YPD plate containing 0.5 mg / mL G418 into the 96-well plate, mix well, and culture at 30°C for 48 hours; take a new sterile 96-well plate and add 190 μL of YPD liquid culture medium to each well. Add 10 μL of the culture obtained from the first 96-well plate to the corresponding wells and culture at 30°C for 24 hours; after 24 hours, take a new sterile 96-well plate and add 190 μL of YPD liquid culture medium to each well. Add 10 μL of the culture obtained from the second 96-well plate to the corresponding wells and culture at 30°C for 24 hours; after 24 hours, take 1 μL from the third 96-well plate and spot it on the YPD plate containing 1.0 mg / mL and 4 mg / mL G418, and continue to culture at 30°C for 96 hours to 120 hours. If the Pichia transformant can grow on a plate containing a high concentration of G418, it means that the transformant fragment has entered the yeast and integrated into the yeast chromosome through homologous recombination. The high-copy recombinant yeast obtained through this step of screening will be more likely to achieve efficient expression of the target protein.

[0049] PCR identification of recombinants: Select a single colony of recombinants and inoculate it into YPD liquid medium, culture it overnight at 30℃ and 220rpm, take 1ml of bacterial solution to extract the genome, and the yeast genomic DNA extraction kit was purchased from Beijing Solebow Technology Co., Ltd. For specific operation steps, refer to the kit instructions. Using genomic DNA as a template, 5'AOX1 and 3'AOX1 universal primers were used as amplification primers for PCR, and the enzyme used was Ex Taq, which was purchased from Beijing Motang Biotechnology Co., Ltd. Among them, the PCR conditions were 98℃ pre-denaturation, 2min, one thermal cycle; 98℃ thermal denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 4min, 25 thermal cycles; 72℃ extension for 2min. The PCR product was subjected to agarose gel electrophoresis. The results showed that the target fragment was successfully amplified and the positive clone was identified.

[0050] Induced expression of recombinant yeast: Select a single colony and place it in a 500mL Erlenmeyer flask containing 10mL BMGY medium. Cultivate at 28-30℃ and 220rpm until OD600 is 2-6. Centrifuge at 1500-3000g for 5min at room temperature to collect the bacteria and resuspend them in BMMY medium to make OD600 about 1. The resulting bacterial solution is placed in a 250mL shake flask and placed on a shaker at 28-30℃ and 220rpm to continue growing for 3 days. Add 100% methanol to the culture medium every 24h to a final concentration of 1.0%.

[0051] 2. Purification of recombinant collagen

[0052] Collect 500 ml of fermentation broth of COL17-CO-1, COL17-CO-2, COL17-CO-3, and COL17-CO-4, centrifuge at 3000g for 20 min at 4°C, and collect the supernatant. After the supernatant is filtered through a 0.22-micron filter membrane, ultrafilter the supernatant collected by centrifugation to 40% of the initial volume, add 5 times the volume of pure water, and then concentrate it to 3% of the initial volume by ultrafiltration. Add 60% saturated ammonium sulfate to the concentrated supernatant, stir for 30 min at room temperature, centrifuge at 9000 rpm for 10 min, collect the precipitate, dissolve the precipitate in 500 mL 0.05 M PBS with a pH of 7.0, and filter it through a 0.22 um filter membrane; ultrafiltration desalting: desalting with a G25 desalting column, that is, use 25 mL of G25 filler, load 5 mL, collect about 10 mL, and desalting can be completed after loading for 10 minutes. Perform SDS-PAGE detection, and the results are as follows Figure 1As shown. The target protein band appeared at the corresponding molecular weight position, and the purity reached 93% to 97% or more. The protein was further concentrated to 2 ml, and the protein concentration was determined by Nanodrop. The final protein concentration was diluted to 100 ml / ml and filled into a freeze-drying bottle at 100 mg. Recombinant human type III collagen was obtained by vacuum freeze drying. The freeze drying parameters were: pre-freezing at -70 for 12 hours, freeze drying at -20℃ for 12 hours, and drying at 20℃ for 14 hours.

[0053] 3. Cell Experiment

[0054] 1. Human dermal fibroblast proliferation experiment

[0055] Human dermal fibroblasts HDFn were inoculated into FM culture medium. After culture to the logarithmic growth phase, HDFn cells in the logarithmic growth phase were inoculated into 96-well plates at a density of 20,000 cells / mL, 100 μL per well. After routine culture for 24 hours, DMEM complete culture medium containing samples was replaced with 100 μL per well. The same volume of DMEM complete culture medium was added to the control group, and the concentration of the sample group (COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ (full-length type Ⅲ recombinant human collagen), COL17 (recombinant human type 17 collagen)) was 5 mg / mL, and 6 replicate wells were set. After culture under saturated humidity for 48 hours, the supernatant was discarded, 10 μL CCK-8 solution was added to each well, and the incubation continued for 2 hours. After the incubation, the OD value was read at 450nm by an enzyme reader to calculate the cell viability. The results are detailed in Figure 2 .

[0056] The results showed that COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, and COLF1 all significantly increased cell viability and promoted cell proliferation. Among them, COL17-CO-2 and COL17-CO-3 were significantly better than the other test groups in promoting cell proliferation.

[0057] 2. COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, and COLF1 promote collagen synthesis. Fibroblasts were cultured at a density of 5×10 5Each well was inoculated into a 24-well plate and cultured for 18 hours. The cells were synchronized by starving with serum-free DMEM medium for 2 hours. 5 mg / mL of COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1 were added, and the sample was cultured for 48 hours, and the cell culture supernatant was collected for use. According to the instructions of the collagen type I, collagen type III, and elastin ELISA kit, the absorbance at 450 nm was measured with an ELISA reader, and the collagen content in the extracellular matrix of HDFn cells was calculated according to the drawn standard curve. The results are shown in Table 2.

[0058] Table 2 The promoting effect of recombinant collagen in this scheme on the synthesis of different collagens and elastin

[0059]

[0060] The results showed that COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17 all significantly promoted the expression of ColI, CoLⅢ, and Col17. Among them, COL17-CO-2 had the best ability to promote the synthesis of type I collagen and elastin, which was significantly better than COLⅢ and COL17. COL17-CO-3 had a better ability to promote the synthesis of COLⅢ, which was significantly better than COLⅢ and COL17.

[0061] 3. Effects of COLFI domain on collagen synthesis and degradation genes

[0062] Skin fibroblasts are one of the main cells that synthesize and secrete collagen, and their secretion and synthesis mechanism is mediated by the TGFβ-Smad signaling pathway. Collagen I, III, IV, and VII are located at the junction of the true epidermis. Increased content can slow down the formation of wrinkles and play an important role in the process of skin aging. Smad3 in the Smad family of proteins plays a key role in the process of TGF-β signaling from cell surface receptors to the cell nucleus. In contrast, Smad7 inhibits the synthesis of collagen mediated by TGFβ1-Smad. As a transforming growth factor, TGFβ1 plays a key role in cell proliferation, differentiation, and death, and regulates the transcriptional synthesis of collagen by activating Smad.

[0063] HDFn cells were inoculated into 12-well plates and cultured for 24 hours. A blank control group (B), a positive control group (PC, 50 ng / mL TGF-β) and a sample group (5 mg / mL COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, COL17) were set up. 2 mL of culture medium was added to each well, and 3 replicates were set up in each group. The cells were placed in an incubator and incubated for 24 hours. RNA was extracted by TRIZOL method, and the expression levels of collagen synthesis-related genes CollagenⅠ, CollagenⅢ, TGFβ1, Smad3, Smad7 and collagen degradation-related genes MMP-1 and MMP-3 were detected by quantitative PCR. The results are detailed in the table. Figure 3 shown.

[0064] The results showed that compared with the BC group, when 5 mg / mL COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17 acted on HDFn cells, each sample group showed a certain promotion of the expression of genes related to collagen synthesis, while reducing the expression of genes related to collagen degradation. Among them, compared with other sample groups, the COL17-CO-2 group was the most significant in increasing the expression of CollagenⅠ gene and TGFβ1 gene. COL17-CO-4 significantly increased the expression of COL17, and had a more significant ability to increase COL17 than other control groups, and had the best ability to reduce MMP-3 and MMP-9. COL17-CO-3 promoted the expression of COLⅢ, Smad3, and Smad7 genes most significantly. COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17 all showed the effect of reducing MMP-3 and MMP-9, among which COL17-CO-2 and COL17-CO-3 had the best effects.

[0065] 4. Research on anti-aging effects of skin cells

[0066] HDFn cells were digested and divided into 6-well plates at a density of 1×106. After culturing for 12 hours, DMEM culture medium containing 5 mg / mL COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17 was added. After culturing for 48 hours, the cell culture medium was removed, washed once with PBS, and 1 ml of staining fixative (4% paraformaldehyde) was added and fixed at room temperature for 15 minutes. The fixative was removed and the cells were washed 3 times with PBS for 3 minutes each time. The PBS was removed and 1 ml of SA-β-gal staining solution was added to each well. Incubate at 37°C overnight and observe and count under an ordinary optical microscope. The results are shown in Table 3.

[0067] Table 3 Anti-aging effect of recombinant collagen in this scheme on skin cells

[0068] Test object Percentage of positive cells SD Control group 32.09 4.23 COL17-CO-1 17.05 1.72 COL17-CO-2 7.59 3.65 COL17-CO-3 8.8 1.03 COL17-CO-4 15.48 2.08 COLF1 18.7 3.33 COLⅢ 24.5 6.61 COL17 20.75 6.08

[0069] The experimental results showed that the number of SA-β-gal stained cells in all test groups was significantly less than that in the control group, indicating that the test substances all had anti-cell aging ability. Among them, the COL17-CO-2 and COL17-CO-3 test groups had the least SA-β-gal positive cells, indicating that they had the best anti-cell aging ability.

[0070] 5. Analysis of the antioxidant effect of the composition

[0071] Skin fibroblasts were seeded in 96-well plates at 20,000 cells per well. After culturing for 24 hours, the original culture medium was carefully aspirated, washed 3 times with PBS, and protected from light damage for 2 hours by adding 400 μmol / L hydrogen peroxide. After washing 3 times with PBS, 100 μL of culture medium containing 5 mg / mL COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17 was added to the cell wells. Each group had 6 replicate wells and was incubated in an incubator for 48 hours. The absorbance (OD) was measured at 450 nm by the CCK-8 method to calculate the cell survival rate. The results are detailed in Figure 4 .

[0072] The results showed that COLⅢ, COLFI, COLFI-1 or COLFI-2 combined with seaweed oligosaccharides could effectively alleviate cell death caused by cell oxidation. COL17-CO-2 and COL17-CO-3 had the best oxidation ability.

[0073] HDFn cells were digested and divided into 6-well plates at a density of 1×106. After culturing for 12 hours, DMEM medium containing 5 mg / mL COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17 was added. After culturing for 48 hours, the cell culture medium was removed and the cells were washed three times with serum-free cell culture medium. The cells were digested with 0.25% trypsin, and after 5 minutes, the cells were observed to fall off under a microscope. The cells were collected in a 15 ml centrifuge tube and centrifuged at 2000 rpm at 4 degrees Celsius for 10 minutes. The positive control cells were suspended in 1 ml of serum-free culture medium. The reactive oxygen positive control (Rosup, 50 mg / ml) was diluted with serum-free culture medium at 1:1000 to a final concentration of 10 micromol / L. The diluted reactive oxygen control was added to the positive control cells and stimulated for 20 minutes. COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, COL17 and untreated cells were collected and suspended in diluted DCFH-DA. The cell concentration was 1 million to 20 million / ml and incubated in a 37℃ cell culture incubator for 20 minutes. Invert and mix every 3-5 minutes to ensure full contact between the probe and the cells. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells. For details, see Figure 5 .

[0074] The results showed that COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17 all have the ability to reduce cellular reactive oxygen species, among which COL17-CO-2 has the most significant ability to inhibit cellular reactive oxygen species.

[0075] 6. Analysis of anti-inflammatory effects

[0076] After culturing RAW246.7 cells on the plate for 24 hours, carefully remove the original culture medium, wash 3 times with PBS, and divide the cells into 96-well plates at 10,000 cells per well. After culturing for 8 hours, remove the supernatant, prepare 100 μL of complete culture medium containing 1 μg / mL LPS and 5 mg / ml concentration of COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17, and add them to the cell wells. Set up 6 replicates for each group, set up a control group with only LPS added, and a control group without LPS added. After incubation in the incubator for 8 hours, carefully remove the supernatant for ELISA analysis of IL-1β and TNF-a analysis. The results are detailed in the table. Figure 6 .

[0077] The results showed that COL17-CO-1, COL17-CO-2, COL17-CO-3, COL17-CO-4, COLF1, COLⅢ, and COL17 could significantly reduce the expression of inflammatory factors IL-1β, IL-6, and TNF-a. COL17-CO-2 and COL17-CO-4 had the best anti-inflammatory effect, which significantly inhibited the expression of IL-1β, TNF-a, and IL-6.

[0078] 4. Effects of COL17-CO-2 on hair follicle cells

[0079] Isolation of pig hair follicles: Take a fresh pig ear, rinse it with tap water for 1 minute, and rinse it twice with 75% ethanol. Cut the skin about 5mm along the edge of the auricle, wash it in PBS containing 1000U / mL penicillin and 1000ug / mL streptomycin for 10 minutes, and put it into Williams E culture medium. Cut it into small pieces of skin with a thickness of about 1.5mm along the direction of hair growth. Under a dissecting microscope, separate the growing follicles, remove the part above the level of the sebaceous gland opening, put the follicles into a 24-well plate, put one follicle in each well, and add 1mL Williams E culture medium for culture. Containing L-glutamine 2.0mmol / L, penicillin 100U / mL and streptomycin 100μg / mL, put it in an incubator at 37℃ with 5% CO2 for culture. There are 10 growing follicles in each experimental group. In the control group, 0.5 mM minoxidil was added to the Williams E medium; in the experimental group, 0.5% COL17-CO-2 recombinant human collagen was added to the Williams E medium. On the first and seventh days of culture, the length from the base of the hair follicle to the top of the hair was measured under an inverted microscope using an eyepiece micrometer. The two measurement results were subtracted to obtain the hair growth length, and the morphological changes of the hair bulb were observed at the same time.

[0080] TUNEL method to detect apoptotic cells in hair follicles: After collecting hair follicles on the 7th day of culture, embed them in OCT, prepare frozen sections with a thickness of 7 μm, fix them with 4% paraformaldehyde at room temperature for 20 min, and follow the instructions of the in situ cell apoptosis detection kit for operation. Counterstain with hematoxylin, dehydrate, make them transparent, seal the sections, and count the number of apoptotic cells. For details, see Figure 7 .

[0081] The results showed that on the 7th day of culture, the average hair growth length of the control group and the experimental group was (2.10±0.03) mm and (2.453±0.03) mm, respectively, with significant differences. On the 7th day of culture, the hair follicles in the control group had undergone degenerative changes, with thinning of the dermal sheath and outer root sheath of the hair follicles, shortening of the hair follicles, and atrophy of the hair bulb; under the action of COL17-CO-2 protein, the hair follicles in the experimental group still showed the morphology of the growing period hair follicles, with little change in the morphological structure of the hair follicles, and the hair matrix cells wrapped the hair papilla. The TUNEL method was used to detect apoptotic cells in the hair follicles, showing that on the 7th day of culture, the average number of apoptotic cells in each hair follicle in the control group and the COL17-CO-2 experimental group was 16.4±0.58 and 7.2±0.41, respectively. The number of apoptotic cells in the hair follicles in the control group was higher than that in the experimental group, with significant differences between the two. The COL17-CO-2 experimental group had the best effect in promoting the average hair growth of hair follicles.

[0082] 5. Effects of COL17-CO-3 on animal skin aging

[0083] 1. Twenty nude mice (19±1) g were selected and randomly divided into 4 groups (blank group, model group, experimental group). The blank group was not irradiated with UVB and was smeared with matrix; the model group was irradiated with UVB and subcutaneously injected with D-galactose (D-GAL) for modeling, and was smeared with matrix; the experimental group was irradiated with UVB and subcutaneously injected with D-GAL for modeling, and was smeared with matrix mixed with COL17-CO-3 mixture. 4 hours after modeling, the matrix was applied to the back of the mice according to the grouping, and 120 μL / mouse was evenly applied on the exposed skin on the back of the mice once a day for 20 days. The UVB irradiation dose was 1 MED in the first week, and then increased by 0.6 MED every week. After the experiment, the mice were killed and the skin tissue on the back of the mice was taken for subsequent experiments. The results are detailed in the literature. Figure 8 .

[0084] After modeling, the epidermal thickness of the model group increased significantly. Compared with the blank group, the cell layer and stratum corneum in the epidermis of the model group became significantly thicker, indicating that the skin barrier was damaged after modeling. The epidermis of the model group was significantly thicker than that of the blank group. After using COL17-CO-3, the degree of proliferation and thickening of epidermal cells and the degree of keratinization were significantly reduced, showing a good resistance to skin damage caused by UVB combined with subcutaneous injection of D-GAL, and the effect was better than that of the positive group.

[0085] 2. Masson staining analysis results are detailed in Fig. 9The results showed that the collagen fibers were sky blue, the collagen fibers in the blank group were arranged regularly and tightly, and the collagen fibers in the model group were curled and loose. After using COL17-CO-3, the expression of collagen fibers increased significantly, and was better than the blank group, and the collagen fibers were arranged more regularly and tightly than the model group. This shows that the use of COL17-CO-3 has a good repair effect on the loss of skin collagen caused by UVB combined with D-GAL subcutaneously.

[0086] 3. Take the remaining back skin, cut off the subcutaneous tissue and weigh it, rinse it in 4℃ 0.9% NaCl solution, wipe it dry with absorbent paper, cut it into pieces, add 4℃ protein lysis solution mixed with protease inhibitor (PMSF), homogenize it with a tissue homogenizer, centrifuge it at 10000r / min for 10min, take out the supernatant, repeat it 3 times, determine the protein content of the supernatant according to the BCA protein quantification kit, and dilute the concentration to the same level. The supernatant was tested and calculated according to the ELISA kit instructions for the content of IL-1, IL-6, TNF-α, MMP3, MMP9 and β-galactosidase in the skin tissue. The results are detailed in Fig.10 .

[0087] Compared with the blank group, the expression levels of IL-1, IL-6, and TNF-α in the model group were significantly increased. After using COL17-CO-3, the skin inflammation caused by UVB combined with D-GAL subcutaneous injection was significantly relieved. The determination of MMP3 and MMP9 in mouse skin showed that compared with the blank group, the expression of MMP3 and MMP9 in the model group was significantly upregulated. Compared with the model group, the use of COL17-CO-3 could significantly downregulate the expression of MMP3 and MMP9 in mouse skin, indicating that COL17-CO-3 could inhibit the upregulation of MMP3 and MMP9 caused by UVB combined with D-GAL subcutaneous injection. The results of β-galactosidase detection in mouse skin showed that the expression of β-galactosidase in the model group was significantly increased compared with the blank group (P<0.05); after using COL17-CO-3, the upregulation of β-galactosidase expression associated with aging caused by UVB combined with D-GAL subcutaneous injection could be alleviated.

[0088] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on its content, and the specific implementation methods and other records in the specification can be used for interpretation.

Claims

1. A novel recombinant collagen, characterized in that: Any one of the recombinant COL17-CO-1, COL17-CO-2, COL17-CO-3 or COL17-CO-4 comprising the functional region of human type XVII collagen and the region 1232 to 1466 of human type III collagen, wherein the amino acid sequences of the functional regions in COL17-CO-1, COL17-CO-2, COL17-CO-3 and COL17-CO-4 are SEQ ID No. 2, SEQ ID No. 6, SEQ ID No. 8 and SEQ ID No. 10, respectively; The amino acid sequence of the region 1232 to 1466 of the human type III collagen is SEQ ID No. 3; There is also a linker between the human type XVII collagen functional region and the human type III collagen 1232-1466 region, the amino acid sequence of the linker is SEQ ID No.4, and the amino acid sequence of the recombinant collagen obtained by recombinant is any one of SEQ ID No.1, SEQ ID No.5, SEQ ID No.7 or SEQ ID No.

9.

2. A composition, characterized in that: It comprises a combination of any one of the recombinants COL17-CO-1, COL17-CO-2, COL17-CO-3, and COL17-CO-4 described in claim 1 and seaweed oligosaccharides.

3. A nucleic acid molecule, characterized in that: It comprises a nucleic acid molecule encoding any one of the recombinant collagen proteins COL17-CO-1, COL17-CO-2, COL17-CO-3 or COL17-CO-4 according to claim 1, and the nucleotide sequences of the nucleic acid molecules encoding the recombinant collagen proteins COL17-CO-1, COL17-CO-2, COL17-CO-3 or COL17-CO-4 are SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, and SEQ ID No.14, respectively.

4. An expression cassette, characterized in that: Comprising the nucleic acid molecule of claim 3.

5. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 3.

6. An expression system, characterized in that: The invention comprises a host cell, wherein the host cell comprises any one of the recombinant collagen according to claim 1, the nucleic acid molecule according to claim 3, the expression cassette according to claim 4 or the expression vector according to claim 5.

7. A novel use of recombinant collagen, characterized in that: The invention relates to use of any one of the recombinant collagen described in claim 1 and the composition described in claim 2 in the preparation of anti-aging drugs.

8. A novel use of recombinant collagen, characterized in that: The invention relates to a method for preparing any one of the recombinant collagen described in claim 1 and the composition described in claim 2 in the preparation of any one of anti-aging cosmetic preparations, daily chemical preparations, medical cosmetology, medical devices or biopharmaceutical preparations.

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