A recombinant type III collagen COLFI domain and its application

Abstract

The present invention relates to genetic engineering, protein engineering technology and its application field, discloses a recombinant type III collagen COLFI domain and its application, the COLFI domain COLFI includes human type III collagen 1232-1466 region, the amino acid sequence of the region is SEQ ID No.1, and the amino acid sequences of the truncated bodies COLFI-1 and COLFI-2 of the COLFI domain are SEQ ID No.2 and SEQ ID No.3 respectively. In the present invention, the human recombinant type III collagen COLFI domain and its truncated body have the function of promoting cell proliferation and migration, the domain and its truncated body can significantly promote the expression of proline hydroxylase (HYP), increase the expression of various collagen synthesis and skin aging related genes such as collagen I, collagen III, collagen XVII, TGFβ1, Smad3, Smad7, etc., and reduce the expression of collagen degradation related bases MMP-1, MMP-3, c-fos, c-jun, etc. And when it is combined with seaweed oligosaccharides, the COLFI domain and its truncations synergistically enhance the effect with seaweed oligosaccharides, which can significantly inhibit skin inflammation and inhibit skin aging.

Description

Technical Field

[0001] The present invention relates to genetic engineering, protein engineering technology and application fields thereof, and in particular to a recombinant type III collagen COLFI domain and application thereof. Background Art

[0002] Inflammation is the body's natural defense mechanism against harmful substances. Usually, the activation of immune cells and other non-immune cells triggers an inflammatory response, promotes tissue repair, and maintains the body's normal functions. Stimulation by certain inflammatory factors or infection by pathogens can trigger an excessive inflammatory response. Excessive inflammatory responses cause deep damage to cells, forming a cycle of inflammatory damage, which leads to further aggravation of the inflammatory response.

[0003] At present, chemical drugs such as trehalose, sodium hyaluronate, chondroitin sulfate, hexanediol, salicylic acid, allantoin, plant extracts, etc. are routinely used to relieve inflammation. However, studies have shown that although chondroitin sulfate has excellent biosafety, it is easy to cause gastrointestinal discomfort. Hexanediol will cause irritation when in contact with the skin for a long time, and it may be corrosive when absorbed through the skin at high concentrations. Sodium hyaluronate will have the risk of excessive inflammation such as rash, itching, erythema, edema, phlebitis, etc. High concentrations of salicylic acid irritate the skin and mucous membranes, have a corrosive effect, and cause a variety of problems such as damaged skin barrier. Allantoin can help the skin resist inflammation, soothe and promote cell repair, but the added dosage is about 0.1%-0.2%. Excessive addition will cause irritation and poor inhibition of inflammation. Some plant extracts, such as aloe extracts and Centella asiatica extracts, have some anti-inflammatory effects in skin care products, but the anti-inflammatory effects are relatively poor, and it is difficult to maintain a good anti-inflammatory effect for a long time. In the field of medical beauty, steroid hormones are also used to inhibit inflammatory responses, but hormone use has strong side effects, and long-term use leads to osteoporosis, hormone disorders and other adverse symptoms. In summary, this field requires an ingredient that is highly safe and can effectively improve skin inflammation.

[0004] Type III collagen is widely used in the field of medical beauty due to its good biological functions. However, the recombinant type III collagen currently used usually adopts the core triple helix structure, which is considered to be the core area of ​​type III collagen for cell adhesion and cell support, and its role in anti-inflammatory is very limited. There is no record of drug development for other parts of type III collagen, resulting in very limited research and development of the structure and function of type III collagen.

[0005] In view of this, the development of a recombinant type III collagen COLFI domain and its application in the preparation of anti-inflammatory, antioxidant and anti-aging drugs can not only effectively make up for the shortcomings of the existing technology, but also further expand the application of recombinant type III collagen in food, cosmetics, medical beauty, health care products, pharmaceuticals and medical devices, etc., which is of great significance for the development of new drugs. Summary of the invention

[0006] The present invention is intended to provide a recombinant type III collagen COLFI domain and application thereof, so as to solve the technical problem that the structural function research and development of type III collagen in the prior art is very limited.

[0007] To achieve the above object, the present invention adopts the following technical scheme: a recombinant type III collagen COLFI domain, including the region 1232-1466 of human type III collagen, the amino acid sequence of which is SEQ ID No.1.

[0008] Preferably, as an improvement, the present scheme also provides a truncation of the COLFI domain of recombinant type III collagen, including truncation I and truncation II, wherein the truncation I (COLFI-1) is the region 1329 to 1466 of human type III collagen, and the amino acid sequence is SEQ ID No. 2; the truncation II (COLFI-2) is the region 1364 to 1466 of human type III collagen, and the amino acid sequence is SEQ ID No. 3.

[0009] Preferably, as an improvement, the present scheme also provides a polypeptide comprising a recombinant type III collagen COLFI domain or a truncate thereof, comprising adding a NotI restriction site (GCGGCCGC) or a histidine tag (HHHHHH) to the 3' end of the COLFI, COLFI-1, COLFI-2 gene sequences to optimize the COLFI, COLFI-1, COLFI-2 gene sequences respectively, the amino acid sequences of COLFI, COLFI-1, COLFI-2 after the optimization by adding the histidine tag are shown in SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8, respectively, and the gene sequences of COLFI, COLFI-1, COLFI-2 after the optimization by adding the NotI restriction site are shown in SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9, respectively.

[0010] Preferably, as an improvement, the present scheme also provides an application of a recombinant type III collagen COLFI domain, including the application of any one of (a) a COLFI domain, (b) a truncation of the COLFI domain, and (c) a polypeptide comprising the COLFI domain or a truncation thereof in the preparation of an anti-inflammatory drug.

[0011] Preferably, as an improvement, the present scheme also provides an application of the COLFI domain of recombinant type III collagen, including the application of any one of (a) the COLFI domain, (b) a truncation of the COLFI domain, and (c) a polypeptide comprising the COLFI domain or a truncation thereof in the preparation of an antioxidant drug.

[0012] Preferably, as an improvement, the present scheme also provides an application of the COLFI domain of recombinant type III collagen, including the application of any one of (a) the COLFI domain, (b) a truncation of the COLFI domain, and (c) a polypeptide comprising the COLFI domain or its truncation in the preparation of anti-aging drugs.

[0013] Preferably, as an improvement, the present scheme also provides a composition comprising human type III collagen RHCⅢ, COLFI, COLFI-1, COLFI-2, or a combination of any one of RHCⅢ, COLFI, COLFI-1, COLFI-2 and seaweed oligosaccharides.

[0014] Preferably, as an improvement, the concentrations of RHCⅢ, COLFI, COLFI-1 and COLFI-2 in the composition are 1-5 mg / ml, and the mass fraction of seaweed oligosaccharides is 0.5-1.5%.

[0015] Preferably, as an improvement, the present solution also provides a use of a composition in the preparation of an anti-inflammatory preparation, an antioxidant preparation or an anti-aging preparation.

[0016] Preferably, as an improvement, the present scheme also provides an application of the COLFI domain of recombinant type III collagen, including (a) the COLFI domain, (b) a truncation of the COLFI domain, (c) a polypeptide comprising the COLFI domain or its truncation, and (d) the application of any raw material in the composition in the preparation of any one of cosmetic preparations, daily chemical preparations, medical beauty preparations, medical device preparations, and biopharmaceutical preparations.

[0017] The principles and advantages of this solution are:

[0018] 1. The present invention has discovered that the COLFI domain of human recombinant type III collagen and its specific truncations have the function of promoting cell proliferation and migration. The domain and its functional truncations can significantly promote the expression of proline hydroxylase (HYP), increase the expression levels of various collagen synthesis and skin aging-related genes such as collagen I, collagen III, collagen XVII, TGFβ1, Smad3, Smad7, etc., and reduce the expression levels of collagen degradation-related genes such as MMP-1, MMP-3, c-fos, c-jun, etc.

[0019] 2. This protocol uses a variety of human recombinant type III collagen fragments, including RHCⅢ, COLFI, COLFI-1, and COLFI-2, and co-cultures them with HSF cells for 24 hours to detect their effects on collagen synthesis and degradation genes. The experiment found that RHCⅢ, COLFI, COLFI-1, and COLFI-2 all have a certain promoting effect on the expression of collagen synthesis genes, and a certain inhibitory effect on the expression of genes related to collagen degradation. Among them, COLFI-1 has a significant function of promoting collagen synthesis and inhibiting collagen degradation, suggesting that COLFI-1 has the best anti-wrinkle effect.

[0020] 3. This scheme found that the combination of COLFI domain and its specific truncations and seaweed oligosaccharides has a synergistic effect in reducing inflammatory responses. The COLFI domain and its specific truncations can significantly enhance the anti-inflammatory effect of seaweed oligosaccharides. The combination has the effect of significantly inhibiting skin inflammation and inhibiting skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the structural prediction of COLF1, COLFI-1 and COLFI-2 based on the RHCⅢCOLFI domain trimer in Example 1 of the present invention (COLF1 on the left, COLF1-1 in the middle, and COLF1-2 on the right).

[0022] Figure 2 The molecular weight electrophoresis diagrams of the COLFI domain of type III collagen (COLFI, one band on the right), COLFI-1 (three bands on the right), and COLFI-2 (two bands on the right) in Experimental Example 1 of the present invention.

[0023] Figure 3 This is a product schematic diagram of the type III collagen COLFI domain (COLFI), COLFI-1, and COLFI-2 in Experimental Example 1 of the present invention.

[0024] Figure 4 This is a comparison chart of the results of the promoting effect of the COLFI domain on HSD proliferation in Experimental Example 2 of the present invention.

[0025] Figure 5 This is a comparison chart of the results of the promotion effect of the COLFI domain on HSF cell migration in Experimental Example 3 of the present invention.

[0026] Figure 6 This is a comparison chart of the results of the COLFI domain promoting collagen synthesis in Experimental Example 4 of the present invention.

[0027] Figure 7 This is a comparison chart of the results of the effect of the COLFI domain on collagen synthesis genes in Experimental Example 5 of the present invention.

[0028] Figure 8 This is a comparison chart of the results of the effect of the COLFI domain on collagen degradation genes in Experimental Example 5 of the present invention.

[0029] Fig. 9 This is a comparison chart of the results of the composition in Experimental Example 7 of the present invention promoting the proliferation of skin fibroblasts.

[0030] Fig.10 This is a comparison chart of the antioxidant effect results of the composition in Experimental Example 9 of the present invention.

[0031] Fig.11 This is a comparison chart of the results of the composition in Experimental Example 10 of the present invention promoting collagen synthesis.

[0032] Fig.12 This is a comparison chart of the results of cytological research on the anti-aging effect of the composition in Experimental Example 11 of the present invention.

[0033] Fig.13 This is a comparison chart of the results of animal studies on the anti-aging effects of the composition in Experimental Example 12 of the present invention (showing changes in IL-1β, IL-6, and TNF-α levels).

[0034] Fig.14 This is a comparison chart of the results of animal studies on the anti-aging effects of the composition in Experimental Example 12 of the present invention (showing the changes in the contents of MMP3 and MMP9).

[0035] Fig.15 This is a comparison chart of the results of the animal study on the anti-aging effect of the composition in Experimental Example 12 of the present invention (showing the changes in the expression level of β-galactosidase). DETAILED DESCRIPTION

[0036] 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.

[0037] Example 1

[0038] This scheme provides a recombinant type III collagen COLFI domain, including the region 1232-1466 of human type III collagen, and the amino acid sequence of the region is SEQ ID No.1.

[0039] The present scheme also provides a truncation of the COLFI domain of recombinant type III collagen, including truncation I and truncation II, wherein truncation I (COLFI-1) is the region 1329 to 1466 of human type III collagen, and the amino acid sequence is SEQ ID No. 2; truncation II (COLFI-2) is the region 1364 to 1466 of human type III collagen, and the amino acid sequence is SEQ ID No. 3.

[0040] The present scheme also provides a polypeptide comprising a recombinant type III collagen COLFI domain or a truncate thereof, comprising adding a NotI restriction site (GCGGCCGC) or a histidine tag (HHHHHH) to the 3' end of the COLFI, COLFI-1, and COLFI-2 gene sequences to optimize the COLFI, COLFI-1, and COLFI-2 gene sequences respectively, wherein the amino acid sequences of COLFI, COLFI-1, and COLFI-2 after the optimization by adding the histidine tag are shown in SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8, respectively, and the gene sequences of COLFI, COLFI-1, and COLFI-2 after the optimization by adding the NotI restriction site are shown in SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9, respectively.

[0041] The gene sequence and amino acid sequence mentioned above are as follows:

[0042] SEQ ID No.1: Amino acid sequence of COLFI domain (region 1232-1466) (COLFI)

[0043] DEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKV

[0044] FCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLD

[0045] VHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYT

[0046] VLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL

[0047] SEQ ID No.2: Amino acid sequence of truncation Ⅰ (region 1329 to 1466) (COLFI-1)

[0048] GESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGN

[0049] VKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIA

[0050] PYDIGGPDQEFGVDVGPVCFL

[0051] SEQ ID No.3: Amino acid sequence of truncation II (region 1364 to 1466) (COLFI-2)

[0052] ASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTK HTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL

[0053] SEQ ID No.4: Amino acid sequence optimized by adding a histidine tag to the 3' end of the COLFI domain (COLFI-HHHHHH)

[0054] DEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLD VHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFLHHHHHH

[0055] SEQ ID No.5: Gene sequence with NotI restriction site added to the 3' end of the COLFI domain (COLFI-GCGGCCGC)

[0056] GATGAGATCATGACTTCATTGAAGTCTGTTAACGGTCAAATCGAATCATTGATCTCACCTGATGGTTCAAGAAAGAACCCAGCTAGAAACTGTAGAGATCTGAAGTTCTGTCATCCAGAACTGAAGTCTGGTGAATACTGGGTTGACCCTAACCAGGGATGTAAGCTGGACGCTATCAAAGTCTTCTGCAACATGGAAACCGGTGAGACTTGTATCTCTGCCAATCCATTGAATGTACCTAGAAAGCATTGGTGGACTGATTCTAGTGCCGAAAAGAAGCATGTTTGGTTCGGTGAAAGTATGGATGGTGGATTCCAGTTCTCTTATGGAAATCCAGAGTTGCCAGAAGATGTGTTGGATGTCCATCTGGCCTTTCTGAGATTGCTGAGTTCTCGTGCTTCTCAGAACATCACCTACCACTGTAAGAACTCTATCGCTTACATGGATCAAGCTAGTGGTAACGTCAAGAAAGCCTTGAAGCTGATGGGTTCTAACGAAGGTGAGTTTAAGGCCGAGGGTAATTCTAAGTTCACCTATACTGTTCTGGAAGACGGTTGTACCAAACACACTGGTGAGTGGTCTAAGACCGTCTTCGAGTACAGAACTCGTAAGGCTGTCAGACTGCCAATTGTTGACATCGCACCTTATGACATTGGTGGTCCAGATCAAGAATTTGGAGTTGATGTTGGACCTGTTTGCTTTCTTCACCACCACCACCACCACTAA GCGGCCGC

[0057] SEQ ID No.6: Optimized amino acid sequence with a histidine tag added to the 3' end of Truncation I (COLFI-1-HHHHHH)

[0058] GESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFLHHHHHH

[0059] SEQ ID No.7: Gene sequence with NotI restriction site added to the 3' end of truncation I (COLFI-1-GCGGCCGC)

[0060] GGTGAAAGTATGGATGGTGGATTCCAGTTTCTTATGGAAATCCAGAGTTGCCAGAAGATGTGTTGGATGTCCATCTGGCCTTTCTGAGATTGCTGAGTTCTCGTGCTTCTCAGAACATCACCTACCACTGTAAGAACTCTATCGCTTACATGGATCAAGCTAGTGGTAACGTCAAGAAAGCCTTGAAGCTGATGGGTTCTAACGAAGGTGAGTTTA AGGCCGAGGGTAATTCTAAGTTCACCTATACTGTTCTGGAAGACGGTTGTACCAAACACACTGGTGAGTGGTCTAAGACCGTCTTCGAGTACAGAACTCGTAAGGCTGTCAGACTGCCAATTGTTGACATCGCACCTTATGACATTGGTGGTCCAGATCAAGAATTTGGAGTTGATGTTGGACCTGTTTGCTTTCTTCACCACCACCACCACCACTAA GCGGCCGC

[0061] SEQ ID No.8: The amino acid sequence optimized by adding a histidine tag to the 3' end of truncation II (COLFI-2-HHHHHH)

[0062] ASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTK HTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFLHHHHHH

[0063] SEQ ID No.9: Gene sequence with NotI restriction site added to the 3' end of truncation II (COLFI-2-GCGGCCGC)

[0064] GCTTCTCAGAACATCACCTACCACTGTAAGAACTCTATCGCTTACATGGATCAAGCTAGTGGTAACGTCAAGAAAGCCTTGAAGCTGATGGGTTCTAACGAAGGTGAGTTTAAGGCCGAGGGTAATTCTAAGTTCACCTATACTGTTCTGGAAGACGGTTGTACCAAACACACTGGTGAGTGGTCTAAGACCGTCTTCGAGTACAGAACTCGTAAGGCTGTCAGACTGCCAATTGTTGACATCGCACCTTATGACATTGGTGGTCCAGATCAAGAATTTGGAGTTGATGTTGGACCTGTTTGCTTTCTTCACCACCACCACCACCACTAA GCGGCCGC

[0065] SEQ ID No.10: Amino acid sequence of human type III collagen (RHCⅢ)

[0066]

[0067] The present invention also provides a composition comprising human type III collagen RHCⅢ, COLFI, COLFI-1, COLFI-2, or a combination of any one of RHCⅢ, COLFI, COLFI-1, COLFI-2 and seaweed oligosaccharides.

[0068] The concentrations of RHCⅢ, COLFI, COLFI-1 and COLFI-2 in the composition are 1-5 mg / ml, and the mass fraction of seaweed oligosaccharide is 0.5-1.5%.

[0069] The present scheme also provides an application of the COLFI domain of recombinant type III collagen, including the application of any one of (a) the COLFI domain, (b) a truncation of the COLFI domain, and (c) a polypeptide comprising the COLFI domain or its truncation in the preparation of an anti-inflammatory drug.

[0070] The present scheme also provides an application of the COLFI domain of recombinant type III collagen, including the application of any one of (a) the COLFI domain, (b) a truncation of the COLFI domain, and (c) a polypeptide comprising the COLFI domain or its truncation in the preparation of an antioxidant drug.

[0071] The present scheme also provides an application of the COLFI domain of recombinant type III collagen, including the application of any one of (a) the COLFI domain, (b) a truncation of the COLFI domain, and (c) a polypeptide comprising the COLFI domain or its truncation in the preparation of anti-aging drugs.

[0072] The scheme also provides use of a composition in the preparation of an anti-inflammatory preparation, an antioxidant preparation or an anti-aging preparation.

[0073] The present scheme also provides an application of the COLFI domain of recombinant type III collagen, including (a) the COLFI domain, (b) a truncation of the COLFI domain, (c) a polypeptide comprising the COLFI domain or its truncation, and (d) the application of any raw material in the composition in the preparation of any one of cosmetic preparations, daily chemical preparations, medical beauty preparations, medical device preparations, and biopharmaceutical preparations.

[0074] Experimental Example 1: Determination of the functional region of the COLFI domain of type III collagen

[0075] The three-dimensional structure of the COLFI domain has been solved, see the PDB database (4AE2). By analyzing the three-dimensional structure of COLFI, it was found that it has a typical trimer structure, which may play an important role in promoting the formation of a trimer structure of type III collagen. After three-dimensional structural analysis, it was found that the GESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSK FTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL (COLFI-1) region or the ASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL (COLFI-2) region of the COLFI domain may independently constitute a functional region. Therefore, after chemical synthesis in the same manner as described above, it was constructed into the pPIC9K vector and expressed.

[0076] (1) Construction of expression vectors for COLFI, COLFI-1, and COLFI-2

[0077] The Pichia pastoris SMD1168 strain and expression vector pPIC9K used in the present invention were purchased from Invitrogen, USA. YPD complete medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose (solid medium containing 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; BMGY medium (yeast growth medium): completely dissolve 10 g yeast extract, 20 g peptone, 3 g K2HPO4, and 11.8 g KH2PO4, and adjust the volume to 890 mL. Autoclave for 20 min, cool to 60°C, add 100 mL of 10×YNB, 1 mL of 500×biotin, and 10 mL of glycerol on a clean bench; BMMY medium: completely dissolve 10 g of yeast extract, 20 g of peptone, 3 g of K2HPO4, and 11.8 g of KH2PO4, and adjust the volume to 895 mL. Autoclave at 121°C for 20 min, cool to 60°C, add 100 mL of 10×YNB, 1 mL of 500×biotin, and 5 mL of methanol on a clean bench.

[0078] The full length of human type III collagen (RHCⅢ) is 1466 amino acids, see (SEQ ID NO.10) for details. According to the gene sequence NM_000090.3 of type III collagen in Genebank, the codon usage frequency of Pichia pastoris was used to optimize the gene codons, eliminate the codons with low usage rate, and add NotI (GCGGCCGC) restriction site or histidine tag (HHHHHH) at the 3' end to optimize the COLFI, COLFI-1, and COLFI-2 gene sequences respectively. The COLFI, COLFI-1, and COLFI-2 gene sequences after adding the NotI restriction site optimization are shown in SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9, respectively. The COLFI, COLFI-1, and COLFI-2 amino acid sequences after adding the histidine tag optimization are shown in SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8, respectively. The optimized COLFI, COLFI-1, and COLFI-2 gene sequences are shown in SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9.

[0079] The optimized gene sequence was synthesized by Qingke Biotechnology Co., Ltd. and directly and seamlessly connected into the NotI site of the pPIC9K vector for sequencing and identification.

[0080] (2) Construction of recombinant Pichia pastoris

[0081] Linearization of expression vectors pPIC9K-His-COLFI, pPIC9K-His-COLFI-1, and pPIC9K-His-COLFI-2: digested with restriction endonuclease SalI at 37°C overnight, the reaction system was as follows: 10 μg of plasmid, 5 μL of 10×H buffer, 50 U of SalI, 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 using a PCR product purification kit (Omega).

[0082] Preparation of Pichia pastoris SMD1168 competent cells: Pick a single colony of yeast SMD1168, inoculate it into a test tube containing 5 mL of YPD liquid culture 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 culture 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; package into 80 μL portions and store at -70°C for later use.

[0083] 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.

[0084] 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.

[0085] 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 (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.

[0086] 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, sealed with double gauze or cheesecloth, and placed on a shaker at 28-30℃ and 220rpm for further growth for 3 days. Add 100% methanol to the culture medium every 24h to a final concentration of 1.0%.

[0087] Collect 500ml of fermentation broth, centrifuge at 3000g for 20min at 4℃, and collect the supernatant. Filter the supernatant through a 0.22μm filter membrane and chromatograph on a nickel column. Elute with 200mmol imidazole. Concentrate 50ml of the eluate to about 2ml, and replace with 10mM tris-HCL PH8.0, 100Mm NaCl. Perform SDS-PAGE detection, the results are as follows Figure 2 As shown. The target protein band appeared at the corresponding molecular weight position, and the purity reached more than 97%. After replacement, the protein was further concentrated to 2ml, and the protein concentration was determined by Nanodrop. The final protein concentration was diluted to 100ml / ml. 100mg was placed in a freeze-drying bottle and vacuum freeze-dried to obtain recombinant human type III collagen. For details, see Figure 3 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.

[0088] Experimental Example 2: Function of the COLFI domain - the promoting effect of the COLFI domain on HSF cell proliferation

[0089] Human skin fibroblasts HSF were cultured to the logarithmic growth phase, the cells were digested and divided into 96-well plates, placed in an incubator (37°C, 5% CO2) for 24 hours, and then the solution was changed and the sample was added. A blank control group (Control) and a sample group were set. The concentration of the sample group was 5 mg / mL, and 6 replicate wells were set. The culture was continued for 48 hours. The supernatant was discarded, and CCK-8 solution was added to each well and incubated for another 2 hours. After the incubation, the OD value was read at 450nm by an enzyme reader to calculate the cell viability.

[0090] Cell activity = OD sample / OD blank × 100%. Figure 4 .

[0091] Figure 4The results showed that after 48 hours of exposure to COLFI and its truncations, 5 mg / mL of COLFI and its truncations significantly promoted the proliferation of human skin fibroblasts. The proliferation effect of COLFI-1 was better than that of RHCⅢ, COLFI-2 or COLFI.

[0092] Experimental Example 3: Function of the COLFI domain: The promoting effect of the COLFI domain on HSF cell migration

[0093] Human fibroblasts were seeded into 6-well plates, 2 mL per well, and placed in a 37°C, 5% CO2 incubator for 18 hours to reach 70%-90% confluence. Then they were washed three times with 200 μL PBS, and a blank control group (BC, serum-free culture medium) and a sample group were set up. The sample group was 5 mg / mL, and 3 replicate wells were set up. The sample group was added with 2 mL of the test sample prepared with serum-free culture medium, and the control group was only added with serum-free culture medium and placed in an incubator (37°C, 5% CO2) for culture. Microscopic observation was performed after 0 and 24 hours, and the scratch area was measured and the cell migration rate was calculated. For details of the results, see Figure 5 .

[0094] Figure 5 The results showed that after cell scratching and culture with RHCⅢ, COLFI, COLFI-1 or COLFI-2 for 24 hours, the migration rate of human skin fibroblasts treated with RHCⅢ, COLFI, COLFI-1 or COLFI-2 was significantly increased compared with the BC group, with significant statistical significance.

[0095] Experimental Example 4: Function of the COLFI domain: The COLFI domain promotes collagen synthesis

[0096] Fibroblasts were cultured at a density of 5 × 10 5 1000 cells / mL were inoculated into 24-well plates and cultured for 24 hours. The cells that were close to confluence were synchronized and starved for 2 hours with serum-free DMEM medium. After sample treatment and culture for 48 hours, the cell culture supernatant was collected for later use. The type 1 collagen ELISA kit was operated according to the instructions, and the absorbance at 450nm was measured with an enzyme reader. The collagen content in the extracellular matrix of HDFn cells was calculated according to the drawn standard curve.

[0097] After culturing HSF cells on the plate for 24 hours, the original culture medium was removed and washed three times with ice PBS. Except for the blank well, the remaining wells were irradiated with 30mJ / cm2 UVB. After washing with PBS three times, 100μL of culture medium containing COLFI, COLFI-1 or COLFI-2 was added to the cell culture wells, with 6 replicates in each group. The HYP content (hydroxyproline, purchased from Nanjing Jiancheng) was determined according to the instructions of the HYPElisa kit. For details, see Figure 6 .

[0098] Figure 6 The results of the HYPElisa experiment showed that the HYP content in the model group was significantly lower than that in the blank group, and COLFI, COLFI-1 or COLFI-2 all significantly upregulated the expression of HYP. COLFI, COLFI-1 or COLFI-2 alleviated the decrease in HYP content caused by UVB irradiation, and the effect of COLFI-1 in repairing HYP expression was significantly better than that of the other experimental groups.

[0099] Experimental Example 5: Function of the COLFI domain - Effects of the COLFI domain on collagen synthesis and degradation genes

[0100] 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.

[0101] HSF 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 RHCⅢ, COLFI, COLFI-1 or COLFI-2) 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, MMP-3, c-fos, c-jun were detected by quantitative PCR. For details, see the results. Figure 7 and Figure 8 .

[0102] Figure 7 Compared with the BC group, when 5 mg / mL RHCⅢ, COLFI, COLFI-1 or COLFI-2 was used to act on human skin fibroblasts, the levels of CollagenⅠ, CollagenⅢ and Smad3 increased significantly, and the level of Smad7 decreased significantly. The above results show that COLFI-1 has a significant function in promoting collagen synthesis.

[0103] Figure 6 The results showed that under UVA stimulation, 5 mg / mL of RHCⅢ, COLFI, COLFI-1 or COLFI-2 could significantly inhibit the expression of collagen degradation-related genes MMP-1, MMP-3, c-jun, and c-fos. COLFI-1 had the best inhibitory effect, suggesting that COLFI-1 has anti-wrinkle effects.

[0104] Experimental Example 6: Analysis of the anti-inflammatory, antioxidant and anti-aging effects of the composition - Optimization of the composition

[0105] Evaluation of the anti-inflammatory effect of each component of the composition: After culturing RAW246.7 cells on the plate for 24 hours, carefully aspirate the original culture medium, wash 3 times with PBS, and divide the cells into 96-well plates at a volume of 10,000 cells per well. After 8 hours of culture, prepare 100 μL of drug-containing culture medium with different mass concentrations in complete culture medium containing 1 μg / mL LPS and add it to different components, with 6 replicates in each group. Set up a control group with only LPS added and a control group without LPS. After incubation in the incubator for 8 hours, carefully aspirate the supernatant for ELISA analysis. The IL-6 concentration was calculated according to the instructions of the kit (Lianke Biotechnology). The results are shown in Table 1:

[0106] Table 1: Effects of different compositions at different concentrations on IL-6 concentration

[0107]

[0108]

[0109] The experimental results show that RHCⅢ, COLFI, COLFI-1 or COLFI-2 can inhibit the production of cellular inflammation. The effect of COLFI, COLFI-1 or COLFI-2 on inflammation is better than that of RHCⅢ, and the inhibitory effect of COLFI-1 is the best, which is about 25% higher than that of full-length RHCⅢ. Seaweed oligosaccharides can also significantly inhibit the expression of inflammatory factor IL-6. At a concentration of 1.5%, the inhibition rate reaches about 50%. In particular, seaweed oligosaccharides have obvious synergistic effects with RHCⅢ, COLFI, COLFI-1 or COLFI-2 in inhibiting inflammation, which significantly enhances the effect of the combination in inhibiting IL-6. Specifically, the synergistic inhibitory effect of COLFI-1+trehalose oligosaccharides is not only better than the IL-6 inhibitory effect when the two are used alone, but also significantly better than the combined effect of D-(+)-trehalose dihydrate or trehalose and COLFI-1. It can be seen that D-(+)-trehalose dihydrate or the combination of trehalose and COLFI-1 has no synergistic effect, but is simply the superposition of the effects of the raw materials.

[0110] Experimental Example 7: Analysis of the anti-inflammatory, anti-oxidative and anti-aging effects of the composition - the promoting effect of the composition on the proliferation of skin fibroblasts

[0111] After digestion of human skin fibroblasts, 2000 cells were allocated to each well of a 96-well plate. After culturing for 24 hours, the original culture medium was carefully aspirated, washed three times with PBS, and 100 μL of RHCⅢ (5 mg / ml); COLFI, COLFI-1 or COLFI-2 and algae oligosaccharide composition (5 mg / ml + 1.5%) was added. There were 6 replicate wells in each group and incubated in an incubator for 48 hours. The absorbance (OD) was measured at 450 nm using the CCK-8 method to calculate the cell survival rate. For details of the results, see Fig. 9 .

[0112] Fig. 9 The results showed that the combination of RHCⅢ, COLFI, COLFI-1 or COLFI-2 and algae oligosaccharide (5 mg / ml+1.5%) significantly promoted the proliferation of skin fibroblasts.

[0113] Experimental Example 8: Analysis of the anti-inflammatory, anti-oxidative and anti-aging effects of the composition - Analysis of the anti-inflammatory effect of the composition

[0114] 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 into different mass concentrations of RHCⅢ, COLFI, COLFI-1 or COLFI-2 and seaweed oligosaccharide mixture (5 mg / ml+1.5%) and add them to the cell wells. Set up 6 duplicate wells 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. The results are shown in Table 2.

[0115] Table 2: Effects of different compositions at different concentrations on IL-1β concentration and TNF-a

[0116] Composition Composition concentration IL-1β concentration (pg / ml) TNF-a (pg / ml) Control(+LPS) - 121.32±4.51 920.84±18.04 RHCⅢ+Algae Oligosaccharide 5mg / ml+1.5% 75.91±5.88 525.96±25.74 RHCⅢ 5mg / ml 109.42±8.30 851.48±10.61 COLFI 5mg / ml 99.76±7.14 812.53±32.76 COLFI-1 5mg / ml 87.53±6.91 756.85±25.74 COLFI-2 5mg / ml 105.3±9.12 825.71±14.13 COLFI+Algae Oligosaccharide 5mg / ml+1.5% 63.75±7.31 715.41±31.53 COLFI-1+Algae Oligosaccharide 5mg / ml+1.5% 51.97±5.67 652.91±41.43 COLFI-2+Algae Oligosaccharide 5mg / ml+1.5% 68.95±4.72 735.65±24.06 Dexamethasone 20 μg / ml 21.54±6.34 398.34±21.54

[0117] The experimental results showed that the mixture of RHCⅢ, COLFI, COLFI-1 or COLFI-2 and seaweed oligosaccharides significantly reduced the expression of inflammatory factors IL-1β and TNF-a compared with trehalose alone and RHCⅢ, COLFI, COLFI-1 or COLFI-2 alone. Among them, the combination of COLFI-1 and seaweed oligosaccharides had the best inhibitory effect.

[0118] Experimental Example 9: Analysis of the anti-inflammatory, anti-oxidative and anti-aging effects of the composition - Analysis of the antioxidant effect of the composition

[0119] 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 HRCⅢ, COLFI, COLFI-1 or COLFI-2 and seaweed oligosaccharide compositions of different mass concentrations were 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. For details of the results, see Fig.10 .

[0120] Fig.10 The results show that the combination of HRCⅢ, COLFI, COLFI-1 or COLFI-2 and seaweed oligosaccharides can effectively alleviate cell death caused by cell oxidation. The combination of COLFI-1 and seaweed oligosaccharides has the best antioxidant capacity.

[0121] Experimental Example 10: Analysis of the anti-inflammatory, anti-oxidative and anti-aging effects of the composition - the effect of the composition on promoting collagen synthesis

[0122] After culturing the cells for 24 hours, the original culture medium was removed and the cells were washed three times with ice PBS. Except for the blank wells, the remaining wells were irradiated with 30mJ / cm2 UVB. After washing with PBS three times, 100μL of culture medium containing COLFI, COLFI-1 or COLFI-2 of different mass concentrations was added to different components, with 6 replicates in each group. The HYP content was determined according to the instructions of the HYPElisa kit (Nanjing Jiancheng). For details, see Fig.11 .

[0123] Fig.11 The results of the HYPElisa experiment showed that the HYP content in the model group was significantly lower than that in the blank group. The mixture of COLFI, COLFI-1 or COLFI-2 and seaweed oligosaccharides all significantly upregulated the expression of HYP (P<0.05), alleviating the decrease in HYP content caused by UVB irradiation. The effect of the COLFI-1 seaweed oligosaccharide mixture in repairing HYP expression was significantly better than that of the other experimental groups (P<0.05).

[0124] Experimental Example 11: Analysis of the anti-inflammatory, anti-oxidative and anti-aging effects of the composition - Cytological study of the anti-aging effect of the composition

[0125] After digestion of skin fibroblasts, cells were divided into 6-well plates. After culturing for 12 hours, COLFI, COLFI-1 or COLFI-2 or a mixture of COLFI, COLFI-1, COLFI-2 and seaweed oligosaccharides were added respectively. After culturing for 48 hours, the cell culture medium was removed, the cells were washed once with PBS or HBSS, 1 ml of staining fixative (4% paraformaldehyde) was added, and the cells were fixed at room temperature for 15 minutes. The fixative was removed, and the cells were washed 3 times with PBS or HBSS for 3 minutes each time. The PBS or HBSS 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. For details of the results, see Fig.12 .

[0126] Fig.12 The results showed that the number of SA-β-gal stained cells in the mixture of COLFI, COLFI-1, COLFI-2 and seaweed oligosaccharides was significantly less than that in the groups with single addition of COLFI, COLFI-1, COLFI-2, RHCⅢ or seaweed oligosaccharides. The number of SA-β-gal stained cells in the complex of COLFI-1 and seaweed oligosaccharides was the least, suggesting that the complex of COLFI-1 and seaweed oligosaccharides has the best anti-cell aging effect.

[0127] Experimental Example 12: Analysis of the anti-inflammatory, anti-oxidative and anti-aging effects of the composition - Zoological evaluation of the anti-aging effect of the composition

[0128] 40 female nude mice (20±1) g were selected and fed adaptively for 5 days. Then, they were numbered and randomly divided into 4 groups (blank group, model group, positive group, COLFI-1 algae oligosaccharide complex group). The blank group was not irradiated with UVB and smeared with matrix; the model group was irradiated with UVB and injected subcutaneously with D-galactose (D-GAL) and smeared with matrix; the positive group was irradiated with UVB and injected subcutaneously with D-GAL and smeared with matrix mixed with vitamin E; the COLFI-1 algae oligosaccharide complex group was irradiated with UVB and injected subcutaneously with D-GAL and smeared with matrix mixed with COLFI-1 algae oligosaccharide (5mg / ml+1.5%). The matrix formula is as follows: Montanov S 7 (7%), glycerol 8 (10%), ethyl paraben (0.1%). 6 hours after modeling, the matrix, positive drugs and were evenly applied on the exposed skin on the back of mice at a rate of 100 μL / mouse once a day for 4 weeks. The mice were killed and the skin tissue on the back of the mice was taken for subsequent testing.

[0129] Cut off the subcutaneous tissue at the smear site 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 (90s each time, repeat 3 times), centrifuge the homogenate at 3000r / min for 15min, take out the supernatant, repeat 3 times, determine the protein according to the BCA protein quantification kit and dilute the protein concentration of the supernatant to the same level. Take an appropriate amount of supernatant and detect and calculate the content of IL-1, IL-6, MMP3, MMP9 and β-galactosidase in skin tissue according to the instructions of the ELISA kit. For details of the results, see Fig.13 , Fig.14 and Fig.15 .

[0130] Fig.13 The results showed that the IL-1β, IL-6, and TNF-α levels in mouse skin were measured by ELISA. The results showed that compared with the blank group, the expression levels of IL-1β and IL-6 in the model group increased significantly. After treatment with vitamin E (1 mg / g) and COLFI-1 seaweed oligosaccharide composite matrix, the levels of the three inflammatory factors decreased significantly (P<0.01).

[0131] Fig.14 The test results of the content 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 (P<0.01); compared with the model group, the COLFI-1 seaweed oligosaccharide composite matrix significantly downregulated the expression of MMP3 in mouse skin (P<0.05) and downregulated the expression of MMP9 (P<0.05).

[0132] Fig.15 The results of the measurement of β-galactosidase content in mouse skin showed that the expression of β-galactosidase in the model group was significantly higher than that in the blank group; after treatment with VE or COLFI-1 seaweed oligosaccharide composite matrix, the β-galactosidase content in mouse skin was significantly downregulated (P<0.05), indicating that COLFI-1 seaweed oligosaccharide composite matrix can effectively alleviate the upregulation of β-galactosidase expression associated with aging. COLFI-1 seaweed oligosaccharide composite matrix has anti-aging function.

[0133] 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, without departing from the technical solution of the present invention, several modifications and improvements can be made, 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 the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A polypeptide of a recombinant type III collagen COLFI domain or a truncated form thereof, characterized in that: The amino acid sequences of the polypeptides are shown in SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.8 respectively.

2. Use of a polypeptide comprising a recombinant type III collagen COLFI domain or a truncate thereof according to claim 1 in the preparation of anti-inflammatory drugs.

3. Use of a polypeptide comprising a recombinant type III collagen COLFI domain or a truncate thereof according to claim 1 in the preparation of an antioxidant drug.

4. Use of a polypeptide comprising a recombinant type III collagen COLFI domain or a truncate thereof according to claim 1 in the preparation of anti-aging drugs.

5. A composition, characterized in that: The invention comprises the polypeptide of the recombinant type III collagen COLFI domain or its truncated form as claimed in claim 1 and human type III collagen RHC III, or a combination of any one of RHC III, recombinant type III collagen COLFI domain or its truncated form polypeptide and seaweed oligosaccharide.

6. A composition according to claim 5, characterized in that: The concentration of the polypeptide of RHC III, recombinant type III collagen COLFI domain or its truncated form in the composition is 1-5 mg / ml, and the mass fraction of seaweed oligosaccharide is 0.5-1.5%.

7. Use of a composition according to claim 6 in the preparation of anti-inflammatory preparations, antioxidant preparations or anti-aging preparations.

8. Use of the polypeptide of the COLFI domain or a truncate thereof according to claim 1 in the preparation of any one of anti-inflammatory, antioxidant, and anti-aging cosmetic preparations, daily chemical product preparations, medical beauty preparations, medical device preparations, and biopharmaceutical preparations.

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