Recombinant sea cucumber collagen with repairing effect and preparation method and application thereof

Abstract

The application relates to the technical field of sea cucumber collagen, in particular to a protein with a repairing effect and a preparation method and application thereof. The amino acid sequence of the protein is from NCBI database collagen [Apostichopus japonicus] GenBank: APA22677.1, is optimized through codons, is further expressed and screened through a shake flask and a 10L fermentation tank, and finally a Pichia sp. strain with a high protein expression amount is found, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 18, 2025, and has a preservation number of CGMCC No. 34894. The obtained pure protein has excellent repairing effect and certain antioxidant effect, and has great commercial value.

Description

Technical Field

[0001] This application relates to the technical field of sea cucumber collagen, specifically to a protein with repairing effects, its preparation method, and its application. Background Technology

[0002] Sea cucumber collagen, extracted from sea cucumbers, is rich in 18 kinds of amino acids. Compared with traditional livestock or fish collagen, it has a higher content of bioactive components and is known as the "gold standard" among collagens. Sea cucumber collagen has excellent anti-aging and repair capabilities, as well as certain antioxidant and anti-inflammatory properties. Currently, all sea cucumber collagen on the market is extracted from sea cucumbers. There are many types of sea cucumbers, among which the Japanese sea cucumber and Dalian sea cucumber, which live at higher latitudes, are considered the best.

[0003] Japanese sea cucumber (Stichopus japonicus), also known as Kanto sea cucumber or Japanese red sea cucumber, is a sea cucumber species with significant economic and nutritional value. Due to its unique biological characteristics and high nutritional value, Japanese sea cucumber has broad application prospects in the food and pharmaceutical industries. Japanese sea cucumber, especially the red sea cucumber from Hokkaido, is considered a premium variety due to its slow growth and high nutritional value, commanding a high market price. High in protein: Japanese sea cucumber is rich in high-quality protein and contains many essential amino acids.

[0004] Currently, sea cucumber collagen is mainly derived from sea cucumbers themselves. However, due to the high price of marine-grown sea cucumbers, a large number of freshwater-farmed sea cucumbers are now cultivated. Sea cucumber collagen is generally extracted from freshwater sea cucumbers, and its nutritional value is far lower than that of marine-grown sea cucumbers. Extracting sea cucumber collagen from marine sea cucumbers results in much higher nutritional value, but the price increases several times over. Furthermore, in recent years, the discharge of nuclear wastewater from Japan has led to marine pollution, posing safety risks to traditionally extracted nutrients from sea cucumbers, including sea cucumber collagen and sea cucumber polysaccharides. Summary of the Invention

[0005] This application addresses the shortcomings of existing methods for obtaining sea cucumber collagen by providing a protein with repairing effects, its preparation method, and its applications. The protein with repairing effects is an amino acid sequence selected from the body wall of the Japanese sea cucumber, as shown in SEQ ID NO. 1. This protein can be obtained in large quantities through genetic engineering techniques, such as constructing recombinant plasmids and transforming them into recipient cells, such as Pichia pastoris cells, followed by culture. Furthermore, this application also yields a recombinant Pichia pastoris strain with high protein expression levels, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 34894. This application ultimately obtains recombinant sea cucumber collagen through green fermentation and purification technologies. This sea cucumber collagen exhibits excellent anti-aging, repair, and antioxidant capabilities, and has significant application value.

[0006] This application involves the following:

[0007] 1. A protein with repair function, having an amino acid sequence as shown in SEQ ID NO.1; or having an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0008] 2. The protein according to claim 1, wherein the nucleotide sequence encoding the protein is as shown in any one of SEQ ID NO. 2 to 4; or has at least 85% sequence identity with any one of SEQ ID NO. 2 to 4.

[0009] 3. A method for preparing the protein with repairing effects described in item 1 or 2, comprising the following steps:

[0010] The nucleotide sequence encoding the protein is recombined into the basic plasmid to obtain the recombinant plasmid;

[0011] Recombinant plasmids are transduced into host cells to obtain genetically engineered cells;

[0012] The genetically engineered cells are cultured, and the protein is isolated from the culture medium.

[0013] 4. The preparation method according to item 3, wherein the basic plasmid is selected from any one or more of the pPIC9K vector, pPICZαA vector, and pHIL-S1 vector;

[0014] And / or, the host cell is selected from any one or more of Pichia pastoris GS115, Pichia pastoris X33, Pichia pastoris MG1003, Pichia pastoris KM71 and Pichia pastoris SMD1168.

[0015] 5. The preparation method according to item 3 or 4, wherein culturing the genetically engineered cells includes seed culture;

[0016] The seed culture medium used in the seed culture is selected from any one or more of BMGY medium, BMMY medium, MGY medium, MGYH medium, RD medium, RDH medium, MD medium, MDH medium, SOC medium and YPD medium.

[0017] And / or, culturing the genetically engineered cells includes fermentation culture;

[0018] The fermentation medium used in the fermentation culture is selected from any one or more of BSM medium, BMMY medium and BMM medium.

[0019] 6. The preparation method according to item 5, wherein the fermentation culture includes methanol induction;

[0020] Methanol induction was initiated when the wet weight of the genetically engineered cells was 100-120 g / L.

[0021] And / or, the methanol concentration during methanol induction is 0.5-1 wt%;

[0022] And / or, the methanol induction time is 80-100 h;

[0023] And / or, methods for separating the protein from the culture medium include any one or more of solid-liquid separation, chromatography, and dialysis;

[0024] Preferably, the solid-liquid separation method includes any one or more of centrifugation, microfiltration, vacuum filtration, and ultrafiltration;

[0025] Preferably, the chromatography includes any one or more of ion chromatography and hydrophobic chromatography.

[0026] 7. A recombinant Pichia pastoris expressing the amino acid sequence shown in SEQ ID NO. 1; and / or,

[0027] It contains nucleotide sequences as shown in any one of SEQ ID NO.2~4.

[0028] 8. A Pichia sp. strain, with accession number CGMCC No. 34894.

[0029] 9. Use of the protein described in item 1- or 2 for promoting skin repair.

[0030] 10. A composition having skin repair efficacy, said composition comprising the protein described in claim 1 or 2, and / or the recombinant Pichia pastoris described in claim 7 or 8.

[0031] 11. The composition according to item 10, wherein the protein content is 0.01 to 2 wt%; preferably 0.05 to 0.8 wt%.

[0032] 12. The antioxidant use of the protein described in item 1 or 2.

[0033] 13. A composition having antioxidant properties, said composition comprising the protein described in claim 1 or 2, and / or the recombinant Pichia pastoris described in claim 7 or 8.

[0034] 14. The composition according to item 13, wherein the protein content is 0.5 to 40 wt%; preferably 1.25 to 20 wt%.

[0035] 15. Use of the protein described in item 1 or 2 in skin care products having skin repair and / or antioxidant effects.

[0036] 16. A repairing emulsion, said emulsion comprising the protein described in claim 1 or 2, and / or the recombinant Pichia sp. described in claim 7, and / or the Pichia sp. described in claim 8;

[0037] Preferably, the protein content is 0.05~5wt%; more preferably 0.1~2wt%.

[0038] 17. A repair dressing comprising the protein described in claim 1 or 2, and / or the recombinant Pichia sp. described in claim 7, and / or the Pichia sp. described in claim 8;

[0039] Preferably, the protein content is 0.05~2wt%.

[0040] 18. A cream dressing, the repair dressing comprising the protein described in claim 1 or 2, and / or the recombinant Pichia sp. described in claim 7, and / or the Pichia sp. described in claim 8;

[0041] Preferably, the protein content is 0.05~2wt%.

[0042] Invention Effects

[0043] 1. The protein provided in this application has significant and excellent skin repair and antioxidant effects.

[0044] 2. This application provides a method for preparing the protein using gene recombination, avoiding safety risks and other issues that exist when extracting the protein from sea cucumbers.

[0045] 3. This application provides a Pichia sp. strain (accession number CGMCC No. 34894) with high expression of this protein, which can be used to achieve large-scale production of this protein. Attached Figure Description

[0046] Figure 1 Electrophoresis images of proteins in various culture media are shown below. Figure 1 As shown, 1-9 represent the protein electrophoresis images in the supernatant after culturing genetically engineered bacteria GS115-1, GS115-2, GS115-3, X33-1, X33-2, X33-3, SMD1168-1, SMD1168-2, and SMD1168-3, respectively.

[0047] Figure 2 BCA standard curve.

[0048] Figure 3 Electrophoretic images of proteins in the culture medium of the genetically engineered bacteria in Comparative Examples 1 and 2, where 1 represents genetically engineered bacteria X33-4 and 2 represents genetically engineered bacteria X33-5.

[0049] Figure 4 . Statistical graph of cell migration rate. Detailed Implementation

[0050] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in their functions.

[0051] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0052] It should be understood that the embodiments of this application described herein include embodiments that are "composed of" and / or "substantially composed of". References to values ​​or parameters of "about" herein include (and describe) variations of that value or parameter itself. For example, a reference to "about X" includes a description of "X".

[0053] As used herein, references to “not” values ​​or parameters generally refer to and describe “except” values ​​or parameters. For example, “The method is not used to treat type X cancer” means that the method is used to treat cancers other than type X.

[0054] As used in this article, the term “approximately XY” has the same meaning as “approximately X to approximately Y”.

[0055] As used herein and in the appended claims, the singular forms “a / an” and “the” include the plural objects unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a preliminary basis for the use of exclusive terms such as “only” or “merely” in conjunction with the description of the elements of the claim, or for the use of the limitation of “no”.

[0056] As used herein, the term "and / or" in words such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in words such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0057] The term "amino acid" and its abbreviations, as commonly understood by those skilled in the art, are as follows: Ala is abbreviated as A, Leu as L, Gln as Q, Ser as S, Arg as R, Lys as K, Glu as E, Thr as T, Asn as N, Met as M, Gly as G, Trp as W, Asp as D, Phe as F, His as H, Tyr as Y, Cys as C, Pro as P, Ile as I, and Val as V.

[0058] A polynucleotide or protein shares a certain percentage of "sequence identity" with another polynucleotide or protein. This means that when aligned, the percentages of bases or amino acids are the same, and the two sequences are in the same relative positions. Sequence identity can be determined in many different ways. To determine sequence identity, various methods and computer programs (such as BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.) can be used to align sequences.

[0059] As used herein, “complementarity” refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid via conventional Watson-Crick base pairing. The complementarity percentage indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., approximately 5, 6, 7, 8, 9, 10 / 10, representing approximately 50%, 60%, 70%, 80%, 90%, and 100% complementarity, respectively). “Complete complementarity” means that all consecutive residues in the nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. As used herein, “substantially complementary” refers to a degree of complementarity of at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of approximately 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or to two nucleic acids hybridizing under stringent conditions.

[0060] As used herein, the term "recombinant" means that a specific nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that produce a construct having a coding or non-coding sequence that can be distinguished from endogenous nucleic acids present in natural systems. "Recombinant nucleic acid," such as a recombinant plasmid, refers to a non-naturally occurring nucleic acid, such as a nucleic acid created through human intervention by artificially combining two separately separated segments of a sequence. This artificial combination is often accomplished by chemical synthesis or by artificially manipulating isolated fragments of nucleic acid. These operations can link nucleic acid fragments with the desired function together to produce the desired functional combination. When recombinant polynucleotides encode polypeptides, the encoded polypeptide sequence can be naturally occurring (wild-type, reference, or standard sequence) or a variant of a naturally occurring polypeptide sequence (e.g., a mutant).

[0061] A “vector” is a composition of substances containing isolated nucleic acids and capable of delivering said isolated nucleic acids into the cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Typically, a suitable vector contains at least one origin of replication functioning in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selective markers. The term “vector” should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc.

[0062] As used herein, the terms “transduction” and “transfection” include methods known in the art for introducing DNA into cells to express a target protein or molecule using infectious agents (such as viruses) or other means. In addition to viral or virus-like reagents, there are chemical-based transfection methods, such as those using calcium phosphate, dendritic polymers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, plasmid delivery, or transposons; particle-based methods, such as those using gene guns, magnetic transfection or magnet-assisted transfection, particle bombardment; and hybridization methods (such as nuclear transfection).

[0063] As used in this article, the terms “transfection,” “transformation,” or “transduction” refer to the process of transferring or introducing exogenous nucleic acids into host cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids.

[0064] As used herein, “variant” is defined as a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains the necessary characteristics. A typical variant of a polynucleotide differs from the nucleic acid sequence of another reference polynucleotide. Changes in the nucleic acid sequence of a variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. Typically, the differences are limited, making the sequences of the reference polypeptide and the variant very similar overall and identical in many regions. The amino acid sequences of the variant and the reference polypeptide can differ by any combination of one or more substitutions, additions, or deletions. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring (such as allelic variants) or may be variants of unknown natural origin. Non-natural variants of polynucleotides and polypeptides can be prepared by mutagenesis, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0065] This application provides a protein with repair efficacy, having the amino acid sequence shown in SEQ ID NO.1; or having an amino acid sequence with at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO.1. In some embodiments, the amino acid sequence of the protein is as shown in SEQ ID NO.1. In some embodiments, the amino acid sequence of the protein has at least 85%, at least 90%, at least 95%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO.1; for example, it may have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO.1. In some embodiments, the protein is a variant of the protein obtained by mutating a conserved sequence of the protein with the amino acid sequence shown in SEQ ID NO.1, thereby having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO.1.

[0066] The protein described in this application is a bioactive sea cucumber collagen protein truncated from the protein in the NCBI database *collagen [Apostichopus japonicus]* ​​GenBank: APA22677.1. Compared to the untruncated protein, the truncated protein has a significantly smaller molecular weight, thus allowing the nucleotide sequence encoding the truncated protein to be recombinated into host cells, such as *Pichia pastoris*, through gene recombination to obtain strains capable of stably and prolifically expressing the truncated protein. In contrast, the original *collagen [Apostichopus japonicus]* ​​GenBank: APA22677.1 protein, due to its excessively large molecular weight, is difficult to stably express in host cells such as *Pichia pastoris*, making it difficult to achieve large-scale production of this protein through gene recombination and the cultivation of recombinant strains.

[0067] In some embodiments, the nucleotide sequence encoding the protein is as shown in any one of SEQ ID NO. 2-4. In some embodiments, the nucleotide sequence encoding the protein has at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of the nucleotide sequences in SEQ ID NO. 2-4; for example, it may have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of the nucleotide sequences shown in SEQ ID NO. 2-4. In some preferred embodiments, the nucleotide sequence encoding the protein is as shown in SEQ ID NO. 2, or is a nucleotide with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO. 2. Experiments have shown that when this nucleotide is recombined into a plasmid and a recombinant strain is further constructed, the expression level of the target protein in the resulting recombinant strain reaches as high as 13.373 g / L, or even as high as 13.5 g / L, 14 g / L, 15 g / L, or even 18 g / L or higher.

[0068] In some embodiments, the protein is natural sea cucumber collagen or recombinant sea cucumber collagen. In some embodiments, the protein is recombinant sea cucumber collagen; it can be used to mass-produce the desired functional sea cucumber collagen through genetic recombination, thereby solving a series of problems associated with natural extraction, such as poor safety and efficacy.

[0069] This application also provides a method for preparing the aforementioned protein with repair effects, which includes the following steps:

[0070] The nucleotide sequence encoding the protein is recombined into the basic plasmid to obtain the recombinant plasmid;

[0071] Recombinant plasmids are transduced into host cells to obtain genetically engineered cells;

[0072] The genetically engineered cells are cultured, and the protein is isolated from the culture medium.

[0073] As those skilled in the art will understand, a basic plasmid is an expression vector for a target gene (i.e., nucleotides encoding a protein). In this application, there are no excessive restrictions on the choice of basic plasmid, as long as it enables the carrying, transduction, and expression of the target gene into the host cell. For example, the basic plasmid can be a pPIC9K vector, a pPICZαA vector, or a pHIL-S1 vector; or any combination of two or more of the above-listed vectors.

[0074] In this application, it is understood that there are no excessive restrictions on the selection of host cells. For example, the host cell may be Pichia pastoris GS115, Pichia pastoris X33, Pichia pastoris MG1003, Pichia pastoris KM71 or Pichia pastoris SMD1168; or any combination of two or more of the above-listed.

[0075] In some embodiments, the seed culture medium used for seed culture is selected from any one or more of BMGY medium, BMMY medium, MGY medium, MGYH medium, RD medium, RDH medium, MD medium, MDH medium, SOC medium, and YPD medium.

[0076] In some implementations, culturing the genetically engineered cells includes fermentation culture.

[0077] In some embodiments, the fermentation medium used during the fermentation culture is selected from any one or more of BSM medium, BMMY medium and BMM medium.

[0078] In some embodiments, the fermentation culture includes methanol induction. In some embodiments, methanol induction is initiated when the wet weight of the genetically engineered cells is 100-120 g / L; for example, methanol induction is initiated when the wet weight of the genetically engineered cells is 100 g / L, 101 g / L, 102 g / L, 103 g / L, 104 g / L, 105 g / L, 106 g / L, 107 g / L, 109 g / L, 110 g / L, 111 g / L, 113 g / L, 114 g / L, 115 g / L, 116 g / L, 117 g / L, 118 g / L, 119 g / L, or 120 g / L. In some embodiments, the methanol concentration during methanol induction is 0.5-1 wt%; for example, the methanol concentration during methanol induction is any concentration within the range of 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, or 0.5-1 wt%. In some embodiments, the methanol induction time is 80-100 h; for example, any time within the range of 80 h, 81 h, 82 h, 83 h, 84 h, 85 h, 86 h, 87 h, 88 h, 89 h, 90 h, 91 h, 92 h, 93 h, 94 h, 95 h, 96 h, 97 h, 98 h, 99 h, 100 h, or 80-100 h.

[0079] In some embodiments, the method for separating the protein from the culture medium includes any one or more of solid-liquid separation, chromatography, and dialysis; the solid-liquid separation described in this application can be performed in a manner conventionally used by those skilled in the art; for example, it can be centrifugation, microfiltration, vacuum filtration, or ultrafiltration. The chromatography described in this application can be performed in a manner conventionally used by those skilled in the art; for example, it can be ion chromatography or hydrophobic chromatography. In some embodiments, the dialysis can be performed in a manner conventionally used by those skilled in the art.

[0080] This application also provides a recombinant Pichia pastoris expressing the amino acid sequence shown in SEQ ID NO. 1. In some embodiments, the recombinant Pichia pastoris comprises the nucleotide sequence shown in any one of SEQ ID NO. 2 to 4.

[0081] This application also provides a Pichia sp., which was deposited on June 18, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101, with accession number: CGMCC No. 34894.

[0082] This application provides a use of the above-mentioned protein in promoting skin repair.

[0083] This application provides a composition with skin repair effects, the composition comprising the above-mentioned protein and / or the above-mentioned recombinant Pichia pastoris.

[0084] In some embodiments, the protein content in the composition is 0.01-2 wt% or 0.05-0.8 wt%; for example, it can be any content within the range of 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, 0.14 wt%, 0.15 wt%, 0.16 wt%, 0.17 wt%, 0.18 wt%, 0.19 wt%, 0.2 wt%, or 0.01-0.2 wt%. In some preferred embodiments, the protein content is 0.05-0.8 wt%; within this range, the composition promotes the migration of human immortalized keratinocytes (HaCaT) by rates as high as 210.3%, or even 220%, 240%, or even 250%.

[0085] This application also provides an antioxidant use of the protein.

[0086] This application provides a composition with antioxidant effects, the composition comprising the above-mentioned protein and / or the above-mentioned recombinant Bipichia pastoris.

[0087] In some embodiments, the protein content in the composition is 0.5-40 wt% or 1.25-20 wt%; for example, any content within the range of 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 22 wt%, 24 wt%, 26 wt%, 28 wt%, 30 wt%, 32 wt%, 34 wt%, 36 wt%, 38 wt%, 40 wt%, or 0.5-40 wt%. In some preferred embodiments, the protein content is 1.25-20 wt%; within this range, the composition exhibits DPPH scavenging rates of 31.52-59.28%, and even as high as 63%, 69%, or even 75%.

[0088] This application also provides the use of the above-mentioned protein in the preparation of skin care products with skin repair and / or antioxidant effects.

[0089] This application also provides an emulsion with repairing effects, the emulsion comprising the above-mentioned protein, and / or the above-mentioned recombinant Pichia pastoris, and / or the above-mentioned Pichia pastoris. Pichia sp. .

[0090] In some embodiments, the protein content is 0.05-5 wt%; preferably 0.1-2 wt%. For example, it can be any content within the range of 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, or 0.05-5 wt%.

[0091] In some embodiments, the emulsion further comprises cosmetically acceptable excipients; for example, these may be thickeners, chelating agents, humectants, emollients, moisturizers, gelling agents, pH adjusters, surfactants, stabilizers, vitamins, penetration enhancers, fragrances, colorants, and solvents, and / or combinations thereof. In some embodiments, the emulsion may also comprise one or more active agents, such as sunscreens, anti-acne agents, anti-microbial agents, anti-wrinkle agents, anti-atrophy agents, anti-inflammatory agents, or optical brighteners. In some embodiments, the aforementioned excipients and / or active agents in the emulsion are added in doses well known to those skilled in the art.

[0092] In some embodiments, the emulsion comprises the following components in parts by weight: 65-75 parts water, 1-10 parts glycerol, 0.5-5 parts 1,2-pentanediol, 0.01-0.5 parts acrylate / C10-30 alkanol acrylate crosspolymer, 1-10 parts Butyrospermum Parkii fruit butter, 0.5-7 parts white beeswax, 0.5-7 parts hydrogenated polyisobutylene, 0.1-5 parts polydimethylsiloxane, 0.5-7 parts caprylic / capric triglyceride, 0.1-5 parts polyglycerol-3-methylglucose distearate, 0.01-3 parts glyceryl stearate / PEG-100 stearate, 0.05-2 parts protein, 0.01-0.7 parts phenoxyethanol / ethylhexylglycerin, and 0.01-0.05 parts sodium hydroxide.

[0093] In some embodiments, the components of the emulsion and the mass percentage of each component are as follows: water balance, glycerol 1-10%, 1,2-pentanediol 0.5-5%, acrylate / C10-30 alkanol acrylate crosspolymer 0.01-0.5%, shea butter (BUTYROSPERMUM PARKII) 1-10%, white beeswax 0.5-7%, hydrogenated polyisobutylene 0.5-7%, polydimethylsiloxane 0.1-5%, caprylic / capric triglyceride 0.5-7%, polyglycerol-3-methylglucose distearate 0.1-5%, glyceryl stearate / PEG-100 stearate 0.01-3%, protein 0.05-2%, phenoxyethanol / ethylhexylglycerin 0.01-0.7%, sodium hydroxide 0.01-0.05%.

[0094] This application also provides a repair dressing comprising the aforementioned protein, and / or the aforementioned recombinant Pichia sp., and / or the aforementioned Pichia sp.

[0095] In some embodiments, the protein content is 0.05~2wt%; for example, it can be any content within the range of 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 2wt%, or 0.05~2wt%.

[0096] In some embodiments, the repair dressing further includes excipients acceptable for medical dressings; for example, these may be thickeners (e.g., xanthan gum), chelating agents (e.g., EDTA), humectants (e.g., citric acid), emollients, moisturizers (e.g., glyceryl glucoside, trehalose, hydroxyethyl cellulose, glycerin, 1,2-hexanediol, etc.), gelling agents, pH adjusters (e.g., citric acid), surfactants, stabilizers, vitamins, film-forming agents (e.g., hydroxyethyl cellulose), penetration enhancers, solvents, and / or combinations thereof. In some embodiments, the repair dressing may also contain one or more active agents, such as antibacterial agents (e.g., citric acid, 1,2-hexanediol, p-hydroxyacetophenone, etc.), anti-wrinkle agents, skin repair agents (e.g., glyceryl glucoside, trehalose, etc.), anti-aging agents (e.g., glyceryl glucoside, etc.), or anti-inflammatory agents (e.g., glyceryl glucoside, citric acid, trehalose). In some embodiments, the above-mentioned excipients and / or active agents in the repair dressing are added in doses well known to those skilled in the art.

[0097] In some embodiments, the repair dressing comprises the following components in parts by weight: 90-100 parts water, 0.01-0.1 parts citric acid, 0.1-1.0 parts trehalose, 1-10 parts 1,3-butanediol, 1-10 parts glycerol, 0.01-0.2 parts hydroxyethyl cellulose, 0.01-0.2 parts xanthan gum, 0.2-1.0 parts p-hydroxyacetophenone, 0.2-1.0 parts 1,2-hexanediol, 0.1-3.0 parts glyceryl glucoside, and 0.05-2 parts protein.

[0098] In some embodiments, the components of the repair dressing and the mass percentage of each component are as follows: water balance, citric acid 0.01-0.1%, trehalose 0.1-1.0%, 1,3-butanediol 1-10%, glycerol 1-10%, hydroxyethyl cellulose 0.01-0.2%, xanthan gum 0.01-0.2%, p-hydroxyacetophenone 0.2-1.0%, 1,2-hexanediol 0.2-1.0%, glyceryl glucoside 0.1-3.0%, and protein 0.05-2%.

[0099] This application also provides a cream dressing comprising the aforementioned protein, and / or the aforementioned recombinant Pichia sp., and / or the aforementioned Pichia sp.

[0100] In some embodiments, the protein content is 0.05~2wt%; for example, it can be any content within the range of 0.01wt%, 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 2wt%, or 0.05~2wt%.

[0101] In some embodiments, the cream dressing further includes excipients acceptable for medical dressings; for example, these may be thickeners (e.g., cetearyl alcohol, hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer, etc.), chelating agents (e.g., EDTA, etc.), humectants (e.g., etc.), emollients (e.g., isononyl isononate, caprylic / capric triglyceride, etc.), defoamers (e.g., dimethicone, etc.), and moisturizers (e.g., glyceryl glucoside, trehalose, hydroxyethyl cellulose, 1,2-pentanediol, glycerin, 1,2-hexanediol, polyglycerol-3-methylglucoside). The dressing may contain glycosaminoglycans, cetearyl alcohol, glyceryl stearate / PEG-100 stearate, cyclopentamethoxysiloxane, caprylic / capric triglyceride, phenoxyethanol / ethylhexylglycerin, etc.), emulsifiers (e.g., cetearyl alcohol, glyceryl stearate / PEG-100 stearate, hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer, etc.), gelling agents, pH adjusters (e.g., citric acid, etc.), surfactants, stabilizers, vitamins, film-forming agents (e.g., hydroxyethyl cellulose, etc.), penetration enhancers, solvents, and / or combinations thereof. In some embodiments, the repair dressing may also contain one or more active agents, such as antibacterial agents (e.g., citric acid, 1,2-hexanediol, p-hydroxyacetophenone, cyclopentamethoxysiloxane, phenoxyethanol / ethylhexylglycerin, etc.), anti-wrinkle agents, skin repair agents (e.g., glyceryl glucoside, trehalose, etc.), anti-aging agents (e.g., glyceryl glucoside, etc.) or anti-inflammatory agents (e.g., glyceryl glucoside, citric acid, trehalose). In some embodiments, the excipients and / or active agents described above in the cream dressing are added in doses known to those skilled in the art.

[0102] In some embodiments, the cream dressing comprises the following components in parts by weight: 70-80 parts water, 2-8 parts glycerin, 0.5-3.0 parts 1,2-pentanediol, 0.1-0.8 parts 1,2-hexanediol, 0.2-1.2 parts glyceryl glucoside, 0.5-3.5 parts polyglycerol-3-methylglucose distearate, 0.5-2.5 parts cetearyl alcohol, 1-3 parts dimethicone, and glyceryl ether. 0.1-1.0 parts by weight of stearate / PEG-100 stearate, 2-6 parts by weight of cyclopentamethoxysiloxane, 1.5-5.5 parts by weight of isononyl isononanoate, 2.0-5.5 parts by weight of caprylic / capric triglyceride, 0.30-0.65 parts by weight of hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer, 0.2-0.8 parts by weight of phenoxyethanol / ethylhexylglycerin, and 0.05-2.00 parts by weight of protein.

[0103] In some embodiments, the components of the cream dressing and the mass percentage of each component are as follows: water balance, glycerin 2-8%, 1,2-pentanediol 0.5-3.0%, 1,2-hexanediol 0.1-0.8%, glyceryl glucoside 0.2-1.2%, polyglycerol-3-methylglucose distearate 0.5-3.5%, cetearyl alcohol 0.5-2.5%, dimethicone 1-3%, glyceryl stearate / PEG-100 stearate 0.1-1.0%, cyclopentamethoxysiloxane 2-6%, isononyl isononanoate 1.5-5.5%, caprylic / capric triglyceride 2.0-5.5%, hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer 0.30-0.65%, phenoxyethanol / ethylhexylglycerin 0.2-0.8%, and protein 0.05-2.00%.

[0104] Example

[0105] Specific embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0106] Example 1. Protein

[0107] The sea cucumber collagen in this application focuses on repair and antioxidant effects. In selecting amino acids, emphasis is placed on repair-promoting active sites (e.g., GED, RGD, GER, GEK, etc.). Furthermore, the arginine (Arg, R), glutamic acid (Glu, E), and lysine (Lys, K) in this sequence are basic amino acids, possessing the ability to scavenge free radicals. Its amino acid sequence is selected from amino acid sequences 708-896 of collagen [Apostichopus japonicus] GenBank: APA22677.1 in the NCBI database, consisting of 195 amino acids. The specific amino acid sequence of this protein is shown in SEQ ID NO.1:

[0108] GEDGDEGRPGPTGPAGPRGSIGPIGPTGITGSPGEKGDQGSPGPAGAQGARGDAGARGANGPAGPQGFPGPAGRPGSPGARGETGPSGIRGETGPAGAAGATGDPGAQGPAGAVGAQGERGEAGNTGPQGPVGNPGIGGPQGNQGPPGPAGQTGPSGAPGAPGERGDPGVAGRDGDQGPVGAPGASGEK.

[0109] The applicant optimized the codons of the nucleotide sequence encoding the protein, naming them nucleotide sequences AS-1, AS-2, and AS-3, respectively. The optimized nucleotide sequences are as follows:

[0110] Nucleotide sequence AS-1, SEQ ID NO.2:

[0111] ;

[0112] Nucleotide sequence AS-2, SEQ ID NO.3:

[0113] GGTGAAGATGGTGACGAGGGCCGACCTGGGCCCACCGGGCCTGCCGGCCCACGAGGCTCTATTGGGCCAATCGGGCCTACAGGTATTACAGGTAGCCCAGGGGAAAAAGGCGACCAGGGCAGTCCTGGACCCGCGGGGGCACAGGGTGCCAGAGGGGATGCTGGTGCGAGGGGAGCTAATGGGCCCGCAGGTCCACAGGGGTTCCCGGGCCCTGCGGGCAGACCAGGTTCCCCTGGTGCCCGCGGCGAGACTGGTCCAAGTGGGATACGGGGAGAGACGGGCCCGGCGGGGGCAGCTGGGGCTACTGGTGACCCAGGAGCACAAGGTCCTGCTGGAGCTGTGGGCGCACAAGGCGAACGCGGAGAGGCCGGTAACACCGGGCCGCAGGGTCCCGTAGGAAATCCGGGAATAGGCGGACCGCAGGGCAACCAAGGGCCGCCAGGCCCCGCAGGCCAAACGGGGCCCTCGGGAGCCCCCGGAGCGCCGGGAGAAAGGGGCGACCCTGGTGTCGCGGGACGTGATGGAGATCAAGGACCGGTTGGTGCCCCCGGAGCTTCAGGGGAAAAG;

[0114] Nucleotide sequence AS-3, SEQ ID NO.4:

[0115] .

[0116] Nucleotide sequences AS-1, AS-2, and AS-3 were recombined into plasmid pPIC9K (Thermo Fisher Scientific v17520) to obtain recombinant plasmids pPIC9K-1, pPIC9K-2, and pPIC9K-3, respectively. Similarly, AS-1, AS-2, and AS-3 were recombined into pPICZαA (Thermo Fisher Scientific v19520) to obtain recombinant plasmids pPICZαA-1, pPICZαA-2, and pPICZαA-3, respectively. Finally, AS-1, AS-2, and AS-3 were recombined into pHIL-S1 (Thermo Fisher Scientific VT1346) to obtain recombinant plasmids pHIL-S1-1, pHIL-S1-2, and pHIL-S1-3, respectively. The construction of all recombinant plasmids was commissioned to Nanjing Ruiyuan Biotechnology, and all samples passed inspection.

[0117] Example 2. Construction of genetically engineered bacteria and screening of single clones

[0118] The specific method is as follows:

[0119] Recombinant plasmids pPIC9K-AS-1, pPIC9K-AS-2, and pPIC9K-AS-3 were electroporated into Pichia pastoris GS115 host cells, respectively, to obtain genetically engineered bacteria GS115-1, GS115-2, and GS115-3, respectively. Similarly, recombinant plasmids pPICZαA-AS-1, pPICZαA-AS-2, and pPICZαA-AS-3 were electroporated into Pichia pastoris X33 host cells, respectively, to obtain genetically engineered bacteria X33-1, X33-2, and X33-3, respectively. Recombinant plasmids pHIL-S1-AS-1, pHIL-S1-AS-2, and pHIL-S1-AS-3 were electroporated into host cells Pichia pastoris SMD1168, respectively, to obtain genetically engineered bacteria SMD1168-1, SMD1168-2, and SMD1168-3, respectively.

[0120] The specific method is as follows: Prepare competent cells of Pichia pastoris GS115, Pichia pastoris X33 and Pichia pastoris SMD1168 respectively, and make their OD600=1.3-1.5. Take 80µl of competent cells, add 6µg of linearized plasmid, and set the electroporation parameters (1.5kV, 25µF, 200Ω). After electroporation, cells were transferred to 2 ml sterile centrifuge tubes and incubated at 30°C for 1-2 hours. Subsequently, resistance selection was performed. Cells electroporated with recombinant plasmids pPIC9K and pHIL-S1 were selected using G418, while cells electroporated with pPICZαA were selected using bleomycin. Resistance selection was then performed according to a gradient of resistance concentrations, and the cells were cultured on YPD plates until single colonies emerged, yielding nine genetically engineered bacteria: GS115-1, GS115-2, GS115-3, X33-1, X33-2, X33-3, SMD1168-1, SMD1168-2, and SMD1168-3.

[0121] Example 3. Preparation of recombinant sea cucumber collagen

[0122] Shake-flask culture for expression and screening:

[0123] 1) Shake flask culture medium formulation

[0124] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, dipotassium hydrogen phosphate 1.8 g / L, potassium dihydrogen phosphate 7.08 g / L, ammonium sulfate 10 g / L, glycerol 12.6 g / L;

[0125] BMMY medium: yeast extract 10g / L, peptone 20g / L, dipotassium hydrogen phosphate 1.8g / L, potassium dihydrogen phosphate 7.08g / L, ammonium sulfate 10g / L, methanol 10ml / L.

[0126] 2) Nine positive clones of different bacterial strains were selected and inoculated into 30 ml of BMGY medium. The cultures were incubated at 29°C and 200 rpm for 2 days. Before induction, the cells were collected by centrifugation at 5000 rpm for 5 min and washed once with water. The cells were then transferred to 30 ml of BMMY medium for methanol-induced expression. Methanol was added to maintain an induction concentration of 0.5-1% per 24 h per flask. After 3 days of induction, the supernatant and precipitate were collected from the shake flasks and analyzed by SDS-PAGE electrophoresis to screen for expression strains.

[0127] Electrophoresis images of proteins in various culture media are shown below. Figure 1 As shown, 1-9 represent the protein electrophoresis images in the supernatant after culturing genetically engineered bacteria GS115-1, GS115-2, GS115-3, X33-1, X33-2, X33-3, SMD1168-1, SMD1168-2, and SMD1168-3, respectively.

[0128] Expression and purification in a 10L fermenter:

[0129] 1) A 10L fermenter, using BSM medium, prepared as follows:

[0130] Fermentation medium: including ammonium dihydrogen phosphate 23.8 g / L, potassium dihydrogen phosphate 5.03 g / L, CaSO4•2H2O 0.59 g / L, K2SO4 9.1 g / L, MgSO4•7H2O 7.45 g / L, and glycerol 40 g / L; after the fermentation medium is sterilized at high temperature, and after the temperature drops to room temperature, add PTM 12.20 ml / L, and adjust the pH to 5.0 with ammonia water.

[0131] Feeding medium: 50% w / v glycerol, with 12 mL PTM1 trace element per liter;

[0132] Induction medium: 100% methanol, with 12 mL of PTM1 trace element per liter.

[0133] 2) Fermentation control: The liquid volume is 5L, the temperature is controlled at 28℃, and the dissolved oxygen is controlled to be greater than 30% by stirring speed and air flow rate. The maximum stirring speed is 800rpm and the maximum air flow rate is controlled at 6L / min.

[0134] 3) Induction: Control the wet weight of the cells to about 100-120 g / L for induction. First starve the cells for 0.5-1 h, then induce with methanol. Add methanol intermittently and control the methanol concentration to 0.5-1% for induction. Induce for 90 h and then transfer to the tank.

[0135] 4) Result determination: After solid-liquid separation, take 5 ml of supernatant and incubate with excess NI FF (NTA) packing material (Lanxiao Technology, A4023205). After washing, elute with 200 mM imidazole. Collect sufficient eluent and then desalt and concentrate using an ultrafiltration concentration tube (Thermo Fisher Scientific, 6 kDa, 50 ml) to control the volume to 1 ml. Finally, use a BCA kit to determine the protein concentration.

[0136] For standard curve preparation, the target protein was purified in the laboratory and prepared according to the concentrations shown in Table 1 below. The absorbance was then measured using a BCA kit (Beyotime P0011) and a microplate reader (Thermo Fisher Multiskan FC A540). The x-axis represents the standard protein concentration, and the y-axis represents the absorbance. The formula is y = 1.8276x + 0.0308. See the detailed standard curve below. Figure 2 The absorbance of the eluent was measured separately, and the protein concentration of the eluent was calculated using the formula x=(y-0.0308) / 1.8276. Three parallel samples were measured for each sample. The average value of the absorbance test results was calculated and then converted into the concentration for the fermenter.

[0137] Table 1. Preparation of BCA reagent standard curve

[0138]

[0139] Table 2. Expression results of different bacterial strains in a 10L fermenter

[0140]

[0141] The protein expression of different bacterial strains is shown in Table 2. Further verification using a 10L fermenter revealed that the genetically engineered strain X33-1 exhibited the best protein expression level. Therefore, genetically engineered strain X33-1 (i.e., Pichia pastoris) was selected as the primary strain. Pichia sp It was deposited on June 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101, with accession number: CGMCC No. 34894.

[0142] purification

[0143] 1) After fermentation, solid-liquid separation was performed. The mixture was centrifuged at 4000 rpm for 30 min and the supernatant was collected.

[0144] 2) Ultrafiltration desalination: Ultrafiltration concentration is performed using an organic membrane with a molecular weight of 6 kDa.

[0145] 3) NTA NI chromatography purification, buffer A: 20mM phosphate buffer + 300mM sodium chloride + 5mM imidazole, pH=7.8; buffer B: 20mM phosphate buffer + 300mM sodium chloride + 500mM imidazole, pH=7.8.

[0146] 4) Ultrafiltration desalination: Ultrafiltration desalination is performed using an organic membrane with a molecular weight of 6 kDa.

[0147] 5) Freeze-drying.

[0148] Comparative Example 1. Protein

[0149] Amino acid sequence 2, whose amino acid sequence is selected from amino acid 35-248 of collagen [Apostichopus japonicus] GenBank: APA22677.1 in the NCBI database, consists of 214 amino acids. The specific amino acid sequence of this protein is shown in SEQ ID NO.5:

[0150] GLTGPKGPKGEPGQTPVIGSGDLIPGPPGPPGGRGGPGDRGPLGPKGNIGIPGPRGGYGADGVPGLPGPPGPPGPPGPSGQLTAEAQVSQQYKGPVMPAGLGYPQAM MAGEPGQRGPPGQAGLRGPAGLTGAPGEPGEQGPTGPVGLTGPAGSPGRPGSAGKDGANGNPGPAGPSGPPGPAGQNGFPGPSGPKGHRGYSGRAGQKGERGDDGER.

[0151] Following the method described in Example 1, the amino acid sequence of the protein described above, such as SEQ ID NO. 5, was codon-optimized and synthesized into the pPICZαA plasmid to obtain the recombinant plasmid pPICZαA-4. Then, following the method described in Example 2, the obtained recombinant plasmid pPICZαA-4 was transformed into the host cell *Pichia pastoris* X33 to obtain the genetically engineered bacterium X33-4. Subsequently, following the method described in Example 3, the obtained genetically engineered bacterium X33-4 was expressed in BMMY medium in shake flasks; and the obtained culture supernatant was subjected to protein electrophoresis, with the results as follows: Figure 3 As shown: 1 represents the protein expression result of the genetically engineered bacterium X33-4 after shaking flask. Its protein expression level is very low and does not meet the requirements for further engineering.

[0152] Comparative Example 2. Protein

[0153] Amino acid sequence 3, whose amino acid sequence is selected from amino acid 441-680 of collagen [Apostichopus japonicus] GenBank: APA22677.1 in the NCBI database, consists of 240 amino acids. The specific amino acid sequence of this protein is shown in SEQ ID NO.6:

[0154] GPQGPAGDRGQLGAPGSIGPAGAAGAKGETGARGGPGSPGSKGSRGEPGRNGDPGLPGTRGLSGNPGKLGKDGSPGPPGPPGKNGRQGATGATGQRGMPGAQGLTGAPGAQGDRGGDGQV GPPGPAGPQGEVGDRGNPGPAGAPGSPGVAGERGAPGLQGPQGQQGPAGLNGPQGESGAPGDAGPQGETGAPGNPGDRGERGGPGDRGPPGPSGRPGGRGVSGRAGQDGATGAPGPKGEQ.

[0155] Following the method in Example 1, the protein with the above-mentioned amino acid sequence, such as SEQ ID NO. 6, was codon-optimized and then synthesized into the pPICZαA plasmid to obtain the recombinant plasmid pPICZαA-5. Then, following the method in Example 2, the obtained recombinant plasmid pPICZαA-5 was transformed into the host cell *Pichia pastoris* X33 to obtain the genetically engineered bacterium X33-5. Subsequently, following the method in Example 3, the obtained genetically engineered bacterium X33-5 was expressed in BMMY medium in shake flasks; and the obtained culture supernatant was subjected to protein electrophoresis, with the results as shown below. Figure 3 As shown: 1 and 2 represent the protein expression results of genetically engineered bacteria X33-4 and X33-5 after shaking flasks, respectively. The protein expression levels of both genetically engineered bacteria are too low and do not meet the requirements for further engineering.

[0156] Example 4. Efficacy Test

[0157] 1. Skin repair effect test

[0158] (1) Test Principle

[0159] Cell migration refers to the movement of cells after receiving migration signals or sensing the effects of certain substances; it is a normal physiological process of growth and development. In human skin, cell migration often occurs during wound healing, similar to the repair function that promotes skin damage healing. Therefore, the migration ability of human keratinocytes can be used to evaluate the impact of a sample on skin repair function to some extent. This experiment established a human immortalized keratinocyte (HaCaT) scratch model and assessed the repair efficacy of the test sample by examining its effect on cell migration.

[0160] (2) Test materials

[0161] ①Testing System

[0162] The cells used in this test were human immortalized keratinocytes (HaCaT), provided by Zhejiang Meisen Cell Technology Co., Ltd.

[0163] ②Main reagents

[0164] DMEM culture medium (Full Gold, FI101-01), fetal bovine serum (Full Gold, FS301-02), trypsin-EDTA (Full Gold, FG301-01), EGF (MCE, HY-P7109), PBS (Biosharp, BL302A).

[0165] ③Main equipment

[0166] CO2 incubator (Shanghai Lishen, HF90), biosafety cabinet (Suzhou Antai, BSC-1304ⅡA2), inverted microscope (Macody, AE2000-T), multi-functional microplate reader (TECAN, INFINITE E PLEX).

[0167] ④ Solution preparation

[0168] Sample solutions: Weigh the sample to be tested and dilute the culture medium to 0.05%, 0.2%, or 0.8% (m / m);

[0169] EGF solution: Weigh a certain amount of EGF powder, add it to PBS to dissolve, and dilute the culture medium to 10 ng / mL before use.

[0170] (3) Test methods

[0171] ① Sample processing and grouping: Based on the cell viability test results, this experiment used 0.05%, 0.2%, and 0.8% (m / m) sample groups, and set up a negative control group and a positive control group (10 ng / mL EGF).

[0172] ② Cell seeding: Draw a straight line across the well on the back of a six-well plate with a marker, then take cells in the logarithmic growth phase, add 2 mL of cell suspension to each well, and incubate in an incubator (37℃, 5% CO2) until the cell confluence reaches 100%.

[0173] ③ Observation and recording: Use a 200μL pipette tip to make a scratch on the back of each well in a straight line perpendicular to the surface. Aspirate the original culture medium, rinse each well twice with an appropriate amount of PBS, add 1mL of PBS and observe under an inverted microscope, making sure the scratch is centered and perpendicular, and take photos to record the results.

[0174] ④ Drug administration: Discard the PBS and add sample solutions of different concentrations, while setting up a negative control group. After drug administration, incubate the 6-well plate in an incubator (37℃, 5% CO2) for 24 hours.

[0175] ⑤ Observation and recording: Discard the original culture medium, rinse each well twice with an appropriate amount of PBS, add 1 mL of PBS under an inverted microscope, observe and photograph at the initial position using the same parameters.

[0176] Cell migration rate = (0h scratch width of sample group - 24h scratch width of sample group) ÷ (0h scratch width of negative control group - 24h scratch width of negative control group) × 100%.

[0177] After testing, referencing Figure 4 The protein obtained by culturing genetically engineered bacteria X33-1 using the method described in Example 3 above showed that it promoted the migration of human immortalized keratinocytes (HaCaT) at concentrations of 0.05%, 0.2%, and 0.8% (m / m), with promotion rates of 37.12%, 180.44%, and 210.3%, respectively. This test evaluated the repair efficacy of the sample by its effect on cell migration after treatment; therefore, the results indicate that the sample has a certain repair efficacy.

[0178] 2. Antioxidant effect

[0179] (1) Test Principle

[0180] 1,1-Diphenyl-trinitrobenzene (DPPH) is a stable, long-lived free radical. Its ethanol solution is deep purple and exhibits strong absorption around 517 nm. In the presence of free radical scavengers, the light absorption of the DPPH ethanol solution decreases due to the pairing of unpaired electrons. The degree of fading of the DPPH ethanol solution is linearly related to the number of electrons it accepts, which can be used to evaluate the sample's ability to scavenge free radicals, i.e., its antioxidant activity.

[0181] (2) Test materials

[0182] ①Main reagents

[0183] DPPH (Maclean), Vitamin E (Maclean), Anhydrous Ethanol (Chinese Medicine).

[0184] ②Main equipment

[0185] Multifunctional microplate reader (TECAN, INFINITE E PLEX), balance (Doris).

[0186] (3) Test methods

[0187] Sample concentration settings

[0188] The samples were diluted with water and mixed to the concentrations shown in Table 3 for later use.

[0189] Table 3 Sample Concentration Design Table

[0190]

[0191] (4) Measurement

[0192] Referring to Table 4, set up sample tubes (T), sample background tubes (T0), DPPH tubes (C), and solvent background tubes (C0). For each sample and each test concentration, three parallel tubes should be set up for each sample tube (T), and three parallel tubes should also be set up for each DPPH tube (C). Add 250 μL of sample solution, 500 μL of sample solvent, and 250 μL of DPPH ethanol solution to the sample tube (T). Add 250 μL of sample solution, 500 μL of sample solvent, and 250 μL of 95% ethanol solution to the sample background tube (T0). Add 750 μL of sample solvent and 250 μL of DPPH ethanol solution to the DPPH tube (C), and add 750 μL of sample solvent and 250 μL of 95% ethanol solution to the solvent background tube (C0). Mix well and let stand at room temperature for 5 min. Add 200 μL of each reaction solution to a 96-well plate and measure the absorbance at 517 nm.

[0193] Table 4 Sample Addition Settings

[0194]

[0195] (5) Calculation formula

[0196]

[0197] In the formula:

[0198] T - Sample absorbance value, which is the absorbance value of the solution after the sample reacts with DPPH;

[0199] T0 - Sample background absorbance value;

[0200] C - Absorbance value of DPPH solution without sample addition;

[0201] C0 - Solvent background absorbance value.

[0202] (6) Sample DPPH removal rate

[0203] The DPPH clearance rate was tested according to the operating procedure. The clearance rates of recombinant sea cucumber collagen at various concentrations are shown in Table 5 below.

[0204] Table 5. DPPH scavenging rate of recombinant sea cucumber collagen at different concentration gradients

[0205]

[0206] According to Table 5, the protein obtained by culturing genetically engineered bacteria X33-1 using the method described in Example 3 above showed DPPH scavenging rates of 31.52%, 37.02%, 47.73%, 50.32%, and 59.28% at concentrations of 1.25–20 wt%, respectively. This test evaluates the antioxidant effect of the sample by detecting its ability to scavenge DPPH free radicals, so the results indicate that the sample has a certain antioxidant effect.

[0207] Example 5. Emulsion

[0208] The protein obtained by culturing genetically engineered bacteria X33-1 using the method described in Example 3 above was used to prepare an emulsion. The emulsion formulation is shown in Table 6.

[0209] Table 6. Emulsion components and their contents

[0210]

[0211] Example 6. Repair Dressing

[0212] The protein obtained by culturing genetically engineered bacteria X33-1 using the method described in Example 3 above was used to prepare a repair dressing. The formula of the repair dressing is shown in Table 7.

[0213] Table 7. Components and Content of Repair Dressing

[0214]

[0215] Example 7. Cream dressing

[0216] The protein obtained by culturing genetically engineered bacteria X33-1 using the method described in Example 3 above was used to prepare a repair dressing. The formula of the repair dressing is shown in Table 8.

[0217] Table 8. Components and Content of Each Component in Repair Dressing

[0218]

[0219] The sequences used in the above embodiments of this application are shown in the following sequence list. It should be understood that the sequences provided in the embodiments of this application are merely exemplary sequences for the embodiments of this application, and not any limitation thereof. The description in this disclosure is given for the purpose of illustration and description, and is not intended to be exhaustive or to limit the disclosure to the disclosed forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of this disclosure, and to enable those skilled in the art to understand this disclosure and design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A protein with repairing effects, the amino acid sequence of which is shown in SEQ ID NO.1; said protein is recombinant sea cucumber collagen.

2. The method for preparing the protein with repair efficacy according to claim 1, comprising the following steps: The nucleotide sequence encoding the protein is recombined into the basic plasmid to obtain the recombinant plasmid; Recombinant plasmids are transduced into host cells to obtain genetically engineered cells; The genetically engineered cells are cultured, and the protein is isolated from the culture medium.

3. The preparation method according to claim 2, wherein, The basic plasmid is selected from any one or more of the pPIC9K vector, pPICZαA vector, and pHIL-S1 vector; And / or, the host cell is selected from any one or more of Pichia pastoris GS115, Pichia pastoris X33, Pichia pastoris MG1003, Pichia pastoris KM71 and Pichia pastoris SMD1168.

4. The preparation method according to claim 2, wherein culturing the genetically engineered cells includes seed culture; The seed culture medium used in the seed culture is selected from any one or more of BMGY medium, BMMY medium, MGY medium, MGYH medium, RD medium, RDH medium, MD medium, MDH medium, SOC medium and YPD medium. And / or, culturing the genetically engineered cells includes fermentation culture; The fermentation medium used in the fermentation culture is selected from any one or more of BSM medium, BMMY medium and BMM medium.

5. The preparation method according to claim 4, wherein the fermentation culture includes methanol induction; in, Methanol induction was initiated when the wet weight of the genetically engineered cells was 100-120 g / L. And / or, the methanol concentration during methanol induction is 0.5-1 wt%; And / or, the methanol induction time is 80-100 h; And / or, methods for separating the protein from the culture medium include any one or more of solid-liquid separation, chromatography, and dialysis.

6. The preparation method according to claim 5, wherein the solid-liquid separation method is selected from any one or more of centrifugation, microfiltration, vacuum filtration, and ultrafiltration.

7. The preparation method according to claim 5, wherein the chromatography is selected from any one or more of ion chromatography and hydrophobic chromatography.

8. A recombinant Pichia pastoris expressing a protein with the amino acid sequence shown in SEQ ID NO. 1; and / or, It contains a sequence encoding a protein as shown in SEQ ID NO.1, wherein the sequence is a nucleotide sequence as shown in any one of SEQ ID NO.2 to 4.

9. A type of Pichia pastoris Pichia sp Its accession number is CGMCC No. 34894.

10. Use of the protein of claim 1 in the preparation of products that promote skin repair.

11. The use of the protein of claim 1 for anti-oxidative purposes in non-disease treatment.

12. Use of the protein of claim 1 in skin care products having skin repair and / or antioxidant effects.

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