Protein with repairing effect as well as preparation method and application thereof

Abstract

The invention relates to the technical field of sea cucumber collagen, in particular to protein with a repairing effect and a preparation method and application thereof. The amino acid sequence of the protein is from Collagen [Apostichopus japonicus] GenBank: APA22677.1 in an NCBI (National Center for Biotechnology Information) database, the strain Pichia sp. With high protein expression quantity is finally found through codon optimization, further expression and screening by virtue of a shake flask and a 10L fermentation tank, the strain Pichia sp. Is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 18, 2025, and the preservation number is CGMCC No.34894. The invention further discloses a preparation method of the Pichia sp. The obtained pure protein has an excellent repairing effect and a certain anti-oxidation effect, and has a great commercial value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sea cucumber collagen protein, and particularly relates to a protein with repair effect and a preparation method and application thereof. BACKGROUND

[0002] Sea cucumber collagen protein is extracted from sea cucumber and rich in 18 kinds of amino acids. Compared with traditional livestock or fish collagen protein, the bioactive ingredients are more abundant, and it is called "the richest in collagen protein". Sea cucumber collagen protein has excellent anti-aging and repair capacity, and also has certain antioxidant and anti-inflammatory capacity. At present, sea cucumber collagen protein on the market is extracted from sea cucumber. There are many kinds of sea cucumber, among which Japanese sea cucumber and Dalian sea cucumber with higher living latitude are the best.

[0003] Japanese sea cucumber (Stichopus japonicus), also known as East Sea sea cucumber and Japanese red sea cucumber, is a sea cucumber variety with important economic value and nutritional value. Japanese sea cucumber has a wide application prospect in the fields of food and medicine due to its unique biological characteristics and high nutritional value. Japanese sea cucumber, especially the red sea cucumber produced in Hokkaido, is considered to be the best sea cucumber because of its slow growth and high nutritional value, and has a high market price. High protein: Japanese sea cucumber is rich in high-quality protein and contains many kinds of amino acids necessary for the human body.

[0004] At present, the source of sea cucumber collagen protein is mainly extracted from sea cucumber. Because sea cucumber grown in the ocean is expensive, there are now a large number of freshwater cultured sea cucumber. Generally, sea cucumber collagen protein is extracted from freshwater sea cucumber, and its nutritional value is much lower than that of sea cucumber grown in the ocean. The nutritional value of sea cucumber collagen protein extracted from marine sea cucumber is very high, but the price will increase several times. In recent years, due to the discharge of nuclear waste water by Japan, the ocean has been polluted. Traditional extraction of nutrients from sea cucumber, including sea cucumber collagen protein and sea cucumber polysaccharide, will have safety risks. SUMMARY

[0005] The present application provides a protein with repair effect and a preparation method and application thereof, aiming at the deficiencies in obtaining sea cucumber collagen protein at present. The protein with repair effect of the present application is a protein selected from the amino acid sequence of Japanese sea cucumber body wall as shown in SEQ ID NO. 1. The protein can be obtained in large quantities by gene engineering technology, such as constructing a recombinant plasmid and transforming the recombinant plasmid into a recipient cell, such as Pichia yeast cell, and then culturing. In addition, the present application also obtains a recombinant Pichia yeast strain with high protein expression, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 34894. The recombinant sea cucumber collagen protein is finally obtained by green fermentation technology and purification technology. The sea cucumber collagen protein has excellent anti-aging and antioxidant capacity, and has good application value.

[0006] The present application relates to the following content:

[0007] 1. A protein having a repairing effect, 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 as shown in SEQ ID NO. 1.

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

[0009] 3. A method for preparing the protein having a repairing effect according to item 1 or 2, comprising the following steps:

[0010] recombining a nucleotide sequence encoding the protein to a basic plasmid to obtain a recombinant plasmid;

[0011] transducing the recombinant plasmid to a host cell to obtain a genetically engineered cell;

[0012] culturing the genetically engineered cell, and separating the protein from the culture solution.

[0013] 4. The method for preparing according to item 3, wherein the basic plasmid is selected from any one or more of pPIC9K vector, pPICZ alpha A 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 method for preparing according to item 3 or 4, wherein culturing the genetically engineered cell comprises seed culture;

[0016] the seed culture medium selected during 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 cell comprises fermentation culture;

[0018] the fermentation culture medium selected during 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 comprises methanol induction.

[0020] wherein the methanol induction is initiated when the wet weight of the genetically engineered cell is 100-120 g / L;

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

[0022] and / or the time of the methanol induction is 80-100 h;

[0023] and / or the method for isolating the protein from the culture solution comprises any one or more of solid-liquid separation, chromatography, and dialysis;

[0024] Preferably, the method for solid-liquid separation comprises any one or more of centrifugation, microfiltration, suction filtration, and ultrafiltration.

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

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

[0027] which comprises a nucleotide sequence as shown in any one of SEQ ID NO. 2-4.

[0028] 8. A Pichia sp. having a preservation number of CGMCC No. 34894.

[0029] 9. Use of the protein according to item 1 or 2 for promoting skin repair.

[0030] 10. A composition having skin repair efficacy, the composition comprising the protein according to item 1 or 2, and / or the recombinant Pichia pastoris according to item 7 or 8.

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

[0032] 12. Use of the protein according to item 1 or 2 for antioxidation.

[0033] 13. A composition having antioxidation efficacy, the composition comprising the protein according to item 1 or 2, and / or the recombinant Pichia pastoris according to item 7 or 8.

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

[0035] 15. Use of the protein according to item 1 or 2 for preparing skin care products having skin repair and / or antioxidant effects.

[0036] 16. An emulsion having repair effects, the emulsion comprising the protein according to item 1 or 2, and / or the recombinant Pichia sp. according to item 7, and / or the Pichia sp. according to item 8.

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

[0038] 17. A repair dressing, the repair dressing comprising the protein according to item 1 or 2, and / or the recombinant Pichia sp. according to item 7, and / or the Pichia sp. according to item 8.

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

[0040] 18. A cream dressing, the repair dressing comprising the protein according to item 1 or 2, and / or the recombinant Pichia sp. according to item 7, and / or the Pichia sp. according to item 8.

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

[0042] Inventive Effects

[0043] 1. The protein provided in the present application has significantly excellent skin repair effects and antioxidant effects.

[0044] 2. The present application provides a method for preparing the protein by gene recombination, which avoids the safety risks and other problems existing in the extraction of the protein from sea cucumbers.

[0045] 3. The present application provides a Pichia sp. (the preservation number is CGMCC No. 34894) having a high expression amount of the protein, and the large-scale production of the protein can be realized by culturing the strain. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The electrophoretic pictures of the proteins in various culture solutions are shown in Figure 1 Fig. 1, wherein 1-9 represent the electrophoretic pictures of the proteins in the supernatant after the culture of the genetic engineering bacteria GS115-1, the genetic engineering bacteria GS115-2, the genetic engineering bacteria GS115-3, the genetic engineering bacteria X33-1, the genetic engineering bacteria X33-2, the genetic engineering bacteria X33-3, the genetic engineering bacteria SMD1168-1, the genetic engineering bacteria SMD1168-2 and the genetic engineering bacteria SMD1168-3, respectively.

[0047] Figure 2 BCA standard curve graph.

[0048] Figure 3 Electrophoresis picture of protein in culture solution of genetically engineered bacteria of Comparative Example 1 and Comparative Example 2, wherein 1 represents genetically engineered bacteria X33-4, and 2 represents genetically engineered bacteria X33-5.

[0049] Figure 4 Cell migration rate histogram. DETAILED DESCRIPTION

[0050] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It is also possible in the art to use different terminology when describing components, alternatives can be used as a matter of engineering choice, and equivalents are considered to be within the scope of the application.

[0051] As used throughout this specification and in the claims, "comprising" or "including" is to be interpreted as "including but not limited to". The description herein is intended to be illustrative only and is presented to enable any person skilled in the art to make and use the application. The present application is not intended to be limited by the embodiments described herein, which are presented as examples of the present application. 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 application pertains. The disclosure of methods and materials herein do not limit their respective scope, but instead, the scope of these methods and materials is to be only limited by the claims.

[0052] It is to be understood that the embodiments of the present application described herein include "consisting" and / or "consisting essentially of embodiments. References to "about" a value or parameter herein include (and describe) variations that are directed to that value or parameter per se. For example, description of "about X" includes description of "X".

[0053] As used herein, references to "not" a value or parameter are generally intended to mean, and are described herein to mean, "except for" the value or parameter. For example, the method is not used to treat cancer of type X means that the method is used to treat cancer other than X.

[0054] As used herein, the term "about X-Y" has the same meaning as "about X to about Y".

[0055] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional element. Thus, the terms "comprising," "including," "containing," etc., are to be construed as open-ended terms (meaning that the composition or process includes, but is not limited to, more than the recited elements), that do not exclude additional, unrecited elements.

[0056] As used herein, the term "and / or," when used in a phrase such as "A and / or B" is intended to mean, in the alternative, A or B; A (alone); or B (alone). Likewise, as used herein, the term "and / or" when used in a phrase such as "A, B, and / or C" is intended to encompass 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," amino acids, and their abbreviations are as generally understood by those skilled in the art and are specifically as follows: Ala is abbreviated A, Leu is abbreviated L, Gin is abbreviated Q, Ser is abbreviated S, Arg is abbreviated R, Lys is abbreviated K, Glu is abbreviated E, Thr is abbreviated T, Asn is abbreviated N, Met is abbreviated M, Gly is abbreviated G, Trp is abbreviated W, Asp is abbreviated D, Phe is abbreviated F, His is abbreviated H, Tyr is abbreviated Y, Cys is abbreviated C, Pro is abbreviated P, He is abbreviated I, Val is abbreviated V.

[0058] A polynucleotide or protein has a certain percent "sequence identity" to another polynucleotide or protein, which means that the percent of bases or amino acids that are the same and in the same relative position when aligned. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.).

[0059] As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid by traditional Watson-Crick base pairing. A percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pair) with a second nucleic acid. "Perfectly complementary" means that all of the contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0060] As used herein, the term "recombinant" means that a specified nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR), and / or ligation steps that result in a construct having a structural coding or non-coding sequence that can be distinguished from endogenous nucleic acids present in natural systems. A "recombinant nucleic acid," such as a recombinant plasmid, refers to a nucleic acid that does not occur naturally, such as a nucleic acid made by the artificial combination of two otherwise separate segments of sequence, by means of human intervention. Such artificial combinations are often accomplished by means of chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid. The above operations can join together nucleic acid segments that have desired functions to produce a desired functional combination. When a recombinant polynucleotide encodes a polypeptide, the encoded polypeptide sequence can be naturally occurring (wild type, parent, or reference sequence) or a variant (e.g., mutant) of a naturally occurring polypeptide sequence.

[0061] A "vector" is a composition of matter comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including but not limited to: linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Typically, a suitable vector will comprise an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction site(s) for cloning, and one or more selectable 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, and the like.

[0062] As used herein, the terms "transduction" and "transfection" include methods known in the art for introducing DNA into cells for expression of a protein or molecule of interest using an infectious agent (such as a virus) or other means. In addition to viral or virus-like agents, there are chemical-based transfection methods, such as transfection using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, plasmid delivery, or transposons; particle-based methods, such as using a gene gun, magnetofection or magnet-assisted transfection, particle bombardment; and hybrid methods (such as nucleofection).

[0063] As used herein, the terms "transfection," "transformation," or "transduction" refer to the process of transferring or introducing foreign nucleic acids into a host cell. A "transfected," "transformed," or "transduced" cell is one that has been transfected, transformed, or transduced with foreign nucleic acid.

[0064] As used herein, "variant" is construed to mean a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleic acid sequence from another, reference polynucleotide. Changes in a variant nucleic acid sequence can or can not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in alterations of an amino acid sequence of a polypeptide encoded by the reference sequence, as set forth below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall. The variant and reference polypeptide can differ in amino acid sequence by as few as one amino acid. The amino acid sequences of the variant and reference polypeptide can differ by one or more substitutions, additions, deletions, fusions and truncations. The substituted, inserted or deleted amino acid residues can or can not be conservative substitutions. Variants of a polynucleotide or polypeptide can be naturally occurring, such as allelic variants, or they can be non-naturally occurring modifications. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those of skill in the art.

[0065] The present application provides a protein having a repairing efficacy, which has an amino acid sequence as set forth in SEQ ID NO. 1; or has an amino acid sequence having at least 85% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1. In some embodiments, the amino acid sequence of the protein is as set forth 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 as set forth in SEQ ID NO. 1; for example, it can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as set forth in SEQ ID NO. 1. In some embodiments, the protein is a variant of the protein having the amino acid sequence as set forth in SEQ ID NO. 1, which is obtained by mutating the conserved sequence of the protein having the amino acid sequence as set forth in SEQ ID NO. 1, whereby it has at least 85% sequence identity to the amino acid sequence as set forth in SEQ ID NO. 1.

[0066] The protein of the present application is a bioactive sea cucumber collagen protein truncated from the protein of collagen [Apostichopus japonicus] GenBank: APA22677.1 in the NCBI database. Compared with the protein before truncation, the molecular weight of the truncated protein is significantly smaller, so that the nucleotide sequence encoding the truncated protein can be recombined into a host cell, such as Pichia pastoris, to obtain a strain capable of stably and highly expressing the truncated protein. The original collagen [Apostichopus japonicus] GenBank: APA22677.1 protein is difficult to stably express in a host cell such as Pichia pastoris due to its large molecular weight, so it is difficult to produce a large amount of the protein by gene recombination and culture of the recombinant strain.

[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 to any one of the nucleotide sequences as shown in SEQ ID NO. 2-4; for example, it can have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the nucleotide sequences as 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 having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence as shown in SEQ ID NO. 2. The present application has found through experiments that the expression amount of the target protein of the recombinant strain obtained by recombining the nucleotide into a plasmid and further constructing a recombinant strain is as high as 13.373 g / L, 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 a natural sea cucumber collagen protein or a genetically recombined sea cucumber collagen protein. In some embodiments, the protein is a genetically recombined sea cucumber collagen protein; it can produce a desired sea cucumber collagen protein with efficacy in a large amount by genetic recombination, to solve a series of problems such as poor safety and efficacy of natural extraction.

[0069] The present application also provides a preparation method of the above-mentioned protein with repair efficacy, which comprises the following steps:

[0070] recombining the nucleotide sequence encoding the protein to a base plasmid to obtain a recombinant plasmid;

[0071] transducing the recombinant plasmid to a host cell to obtain a genetically engineered cell;

[0072] culturing the genetically engineered cell, and isolating the protein from the culture solution.

[0073] As understood by those skilled in the art, the base plasmid is an expression vector for the target gene (i.e. the nucleotide encoding the protein). In the present application, there is no excessive limitation for the selection of the base plasmid, which can realize the carrying, transduction to the host cell and expression of the target gene. Exemplarily, the base plasmid can be a pPIC9K vector, a pPICZ alpha A vector or a pHIL-S1 vector; or a combination of any two or more of the above.

[0074] In the present application, it can be understood that there is no excessive limitation for the selection of the host cell. Exemplarily, the host cell can be Pichia pastoris GS115, Pichia pastoris X33, Pichia pastoris MG1003, Pichia pastoris KM71 or Pichia pastoris SMD1168; or a combination of any two or more of the above.

[0075] In some embodiments, the seed culture medium selected for 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.

[0076] In some embodiments, the culturing of the genetically engineered cell comprises fermentation culture.

[0077] In some embodiments, the fermentation culture medium selected for 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 comprises methanol induction. In some embodiments, the methanol induction is initiated when the wet weight of the genetically engineered cells is 100-120 g / L; for example, the 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 concentration of methanol during the methanol induction is 0.5-1 wt%; for example, the concentration of methanol during the methanol induction is 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, or any concentration within the range of 0.5-1 wt%. In some embodiments, the time of the methanol induction is 80-100 h; for example, 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 any time within the range of 80-100 h.

[0079] In some embodiments, the method of isolating the protein from the culture solution comprises any one or more of solid-liquid separation, chromatography, and dialysis; the solid-liquid separation described herein can be performed in a manner routinely employed by one of skill in the art; for example, it can be centrifugation, microfiltration, suction filtration, or ultrafiltration. The chromatography described herein can be performed in a manner routinely employed by one of skill in the art; for example, it can be ion chromatography or hydrophobic chromatography. In some embodiments, the dialysis can be performed in a manner routinely employed by one of skill in the art.

[0080] The present application also provides a recombinant Pichia sp. expressing an amino acid sequence as shown in SEQ ID NO. 1. In some embodiments, the recombinant Pichia sp. comprises a nucleotide sequence as shown in any one of SEQ ID NO. 2-4.

[0081] The present application also provides a Pichia sp. deposited with the China General Microbiological Culture Collection Center (CGMCC) on June 18, 2025, at the address of No. 1, Yihuangyuan, Beichen West Road, Haidian District, Beijing, China, by the Institute of Microbiology, Chinese Academy of Sciences, with a postal code of 100101, and with a deposit number of CGMCC No. 34894.

[0082] The present application provides a use of the above-mentioned protein for promoting skin repair.

[0083] The present application provides a composition with skin repair effect, which comprises the above-mentioned protein and / or the above-mentioned recombinant Pichia pastoris.

[0084] In some embodiments, the content of the protein in the composition is 0.01-2 wt%, 0.05-0.8 wt%; for example, it can be any content in 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 content of the protein is 0.05-0.8 wt%; within the range, the composition has a promoting effect on the cell migration of human immortalized keratinocytes (HaCaT) with a promoting rate of up to 210.3%, even 220%, 240% or even 250%.

[0085] The present application also provides the use of the protein for antioxidation.

[0086] The present application provides a composition with antioxidation effect, which comprises the above-mentioned protein and / or the above-mentioned recombinant Pichia pastoris.

[0087] In some embodiments, the content of the protein in the composition is 0.5-40 wt%, 1.25-20 wt%; for example, it can be any content in 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 content of the protein is 1.25-20 wt%; within the range, the composition has a DPPH clearance rate of 31.52-59.28%, even up to 63%, 69% or even 75%.

[0088] The present application also provides the use of the above-mentioned protein for preparing skin care products with skin repair and / or antioxidation effect.

[0089] The present application also provides a lotion having repairing efficacy, which comprises the above-mentioned protein, and / or the above-mentioned recombinant Pichia sp., and / or the above-mentioned Pichia sp.

[0090] In some embodiments, the content of the protein 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 lotion further comprises cosmetically acceptable excipients; for example, thickening agents, chelating agents, moisturizing agents, emollients, humectants, gelling agents, pH adjusting agents, surfactants, stabilizers, vitamins, penetration enhancers, fragrances, colorants and solvents, and / or combinations thereof. In some embodiments, the lotion can further comprise one or more active agents, such as sunscreen agents, anti-acne agents, anti-microbial agents, anti-wrinkle agents, anti-atrophy agents, anti-inflammatory agents or optical brighteners. In some embodiments, the above-mentioned excipients and / or active agents in the lotion are added in dosages well known to those skilled in the art.

[0092] In some embodiments, the lotion comprises the following components by weight: water 65-75 parts by weight, glycerin 1-10 parts by weight, 1,2-pentanediol 0.5-5 parts by weight, acrylates / C10-30 alkyl acrylate crosspolymer 0.01-0.5 parts by weight, butyrospermum parkii (shea) butter 1-10 parts by weight, white beeswax 0.5-7 parts by weight, hydrogenated polyisobutene 0.5-7 parts by weight, dimethicone 0.1-5 parts by weight, caprylic / capric triglyceride 0.5-7 parts by weight, polyglyceryl-3 methylglycose distearate 0.1-5 parts by weight, glyceryl stearate / PEG-100 stearate 0.01-3 parts by weight, protein 0.05-2 parts by weight, phenoxyethanol / ethylhexylglycerin 0.01-0.7 parts by weight, sodium hydroxide 0.01-0.05 parts by weight.

[0093] In some embodiments, the components of the emulsion and the mass percentage of each component are as follows: water balance, glycerin 1-10%, 1,2-pentanediol 0.5-5%, acrylates / C10-30 alkyl acrylate crosspolymer 0.01-0.5%, butyrospermum parkii (shea) butter 1-10%, cera alba 0.5-7%, hydrogenated polyisobutene 0.5-7%, dimethicone 0.1-5%, caprylic / capric triglyceride 0.5-7%, polyglyceryl-3 methyl glucose 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] The present application also provides a repair dressing comprising the above-mentioned protein, and / or the above-mentioned recombinant Pichia sp., and / or the above-mentioned Pichia sp.

[0095] In some embodiments, the content of the protein is 0.05-2wt%; for example, it can be 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 any content within the range of 0.05-2wt%.

[0096] In some embodiments, the repair dressing further comprises an excipient acceptable in medical dressings; for example, it can be a thickening agent (such as xanthan gum, etc.), a chelating agent (such as EDTA, etc.), a moisturizing agent (such as citric acid, etc.), an emollient, a humectant (such as glycerol glucoside, trehalose, hydroxyethyl cellulose, glycerol, 1,2-hexanediol, etc.), a gelling agent, a pH regulator (such as citric acid, etc.), a surfactant, a stabilizer, a vitamin, a film-forming agent (such as hydroxyethyl cellulose, etc.), a penetration enhancer, a solvent, and / or a combination thereof. In some embodiments, the repair dressing can further comprise one or more active agents; for example, it can be an antibacterial agent (such as citric acid, 1,2-hexanediol, p-hydroxyacetophenone, etc.), an anti-wrinkle agent, a skin repair agent (such as glycerol glucoside, trehalose, etc.), an anti-aging agent (such as glycerol glucoside, etc.), or an anti-inflammatory agent (such as glycerol glucoside, citric acid, trehalose). In some embodiments, the above-mentioned excipients and / or active agents in the repair dressing are added in a dosage well known to those skilled in the art.

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

[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%, glycerol glucoside 0.1-3.0%, protein 0.05-2%.

[0099] The present application also provides a cream dressing, which comprises the above-mentioned protein, and / or the above-mentioned recombinant Pichia pastoris, and / or the above-mentioned Pichia sp.

[0100] In some embodiments, the content of the protein is 0.05-2 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% or 0.05-2 wt%.

[0101] In some embodiments, the cream dressing further comprises an excipient acceptable in a medical dressing; for example, can be, a thickening agent (e.g., cetostearyl alcohol, hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer, etc.), a chelating agent (e.g., EDTA, etc.), a moisturizing agent (e.g., glycerin, etc.), an emollient (e.g., isononyl isononanoate, caprylic / capric triglyceride, etc.), an antifoaming agent (e.g., dimethicone, etc.), a humectant (e.g., glycerin glucoside, trehalose, hydroxyethyl cellulose, 1,2-pentanediol, glycerin, 1,2-hexanediol, polyglyceryl-3 methyl glucose distearate, cetostearyl alcohol, glyceryl stearate / PEG- 100 stearate, cyclomethicone, caprylic / capric triglyceride, phenoxyethanol / ethylhexylglycerin, etc.), an emulsifier (e.g., cetostearyl alcohol, glyceryl stearate / PEG- 100 stearate, hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer, etc.), a gelling agent, a pH adjuster (e.g., citric acid, etc.), a surfactant, a stabilizer, a vitamin, a film-forming agent (e.g., hydroxyethyl cellulose, etc.), a penetration enhancer, a solvent, and / or a combination thereof. In some embodiments, the protective dressing can further comprise one or more active agents, for example, can be an antibacterial agent (e.g., citric acid, 1,2-hexanediol, p-hydroxyacetophenone, cyclomethicone, phenoxyethanol / ethylhexylglycerin, etc.), an anti-wrinkle agent, a skin repair agent (e.g., glycerin glucoside, trehalose, etc.), an anti-aging agent (e.g., glycerin glucoside, etc.), or an anti-inflammatory agent (e.g., glycerin glucoside, citric acid, trehalose). In some embodiments, the above excipients and / or active agents in the cream dressing are added in dosages well known to those skilled in the art.

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

[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%, polyglyceryl-3 methylglucose distearate 0.5-3.5%, cetostearyl alcohol 0.5-2.5%, dimethicone 1-3%, glyceryl stearate / PEG-100 stearate 0.1-1.0%, cyclopentasiloxane 2-6%, isononyl isononanoate 1.5-5.5%, caprylic / capric triglyceride 2.0-5.5%, hydroxyethyl acrylate / sodium acryloyldimethyl taurate copolymer 0.30-0.65%, phenoxyethanol / ethylhexylglycerin 0.2-0.8%, protein 0.05-2.00%.

[0104] Embodiment

[0105] Specific embodiments of the present application will be described in greater detail herein below with reference to the accompanying drawings. Although specific embodiments of the present application are shown in the drawings, it is understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present application can be more thoroughly understood and the scope of the present application can be accurately conveyed to those skilled in the art.

[0106] Example 1. Protein

[0107] The sea cucumber collagen protein of the present application focuses on repair and antioxidant efficacy, and when selecting amino acids, the active sites for repair (such as GED, RGD, GER, GEK, etc. active sites) are considered, and the sequence arginine (Arg, R), glutamic acid (Glu, E), lysine (Lys, K) is an alkaline amino acid, which has the ability to scavenge free radicals. The amino acid sequence is selected from the 708-896 amino acid sequence in the NCBI database collagen [Apostichopus japonicus] GenBank: APA22677.1, which consists of 195 amino acids. The amino acid sequence of the protein is specifically shown as SEQ ID NO. 1:

[0108] GEDGDEGRPGPTGPAGPRGSIGPIGPTGITGSPGEKGDQGSPGPAGAQGARGDAGARGANGPAGPQGFPGPAGRPGSPGARGETGPSGIRGETGPAGAAGATGDPGAQGPAGAVGAQGERGEAGNTGPQGPVGNPGIGGPQGNQGPPGPAGQTGPSGAPGAPGERGDPGVAGRDGDQGPVGAPGASGEK.

[0109] The nucleotide sequence encoding the protein is codon-optimized by the applicant, and is named as nucleotide sequence AS-1, nucleotide sequence AS-2, and nucleotide sequence AS-3, respectively. The optimized nucleotide sequences are as follows:

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

[0111] GGAGAAGATGGAGATGAGGGAAGACCAGGACCTACCGGACCTGCAGGTCCTAGAGGTTCCATTGGACCCATTGGCCCCACCGGAATCACTGGATCACCCGGAGAAAAGGGAGATCAAGGTTCTCCTGGTCCCGCCGGCGCACAAGGTGCTAGAGGAGATGCAGGTGCTAGGGGAGCTAACGGACCCGCAGGTCCTCAAGGATTTCCAGGCCCAGCAGGCAGGCCAGGTAGTCCTGGAGCAAGAGGTGAGACGGGCCCTAGTGGAATCAGAGGTGAAACGGGCCCAGCCGGAGCTGCCGGAGCAACCGGTGATCCAGGTGCTCAAGGCCCAGCAGGTGCAGTTGGCGCACAGGGAGAACGTGGCGAAGCTGGAAATACCGGTCCACAGGGTCCAGTCGGAAATCCTGGCATCGGTGGTCCTCAAGGAAACCAAGGACCTCCCGGTCCTGCCGGTCAGACTGGTCCTTCTGGTGCACCAGGTGCTCCCGGTGAAAGAGGTGATCCTGGCGTGGCTGGTCGAGATGGAGATCAAGGTCCCGTGGGAGCTCCTGGTGCCTCTGGTGAAAAA;

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

[0113] GGTGAAGATGGTGACGAGGGCCGACCTGGGCCCACCGGGCCTGCCGGCCCACGAGGCTCTATTGGGCCAATCGGGCCTACAGGTATTACAGGTAGCCCAGGGGAAAAAGGCGACCAGGGCAGTCCTGGACCCGCGGGGGCACAGGGTGCCAGAGGGGATGCTGGTGCGAGGGGAGCTAATGGGCCCGCAGGTCCACAGGGGTTCCCGGGCCCTGCGGGCAGACCAGGTTCCCCTGGTGCCCGCGGCGAGACTGGTCCAAGTGGGATACGGGGAGAGACGGGCCCGGCGGGGGCAGCTGGGGCTACTGGTGACCCAGGAGCACAAGGTCCTGCTGGAGCTGTGGGCGCACAAGGCGAACGCGGAGAGGCCGGTAACACCGGGCCGCAGGGTCCCGTAGGAAATCCGGGAATAGGCGGACCGCAGGGCAACCAAGGGCCGCCAGGCCCCGCAGGCCAAACGGGGCCCTCGGGAGCCCCCGGAGCGCCGGGAGAAAGGGGCGACCCTGGTGTCGCGGGACGTGATGGAGATCAAGGACCGGTTGGTGCCCCCGGAGCTTCAGGGGAAAAG;

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

[0115] GGTGAAGATGGTGATGAAGGTAGACCAGGTCCAACTGGTCCAGCTGGTCCAAGAGGTTCTATTGGTCCAATTGGTCCAACTGGTATTACTGGTTCTCCAGGTGAAAAGGGTGATCAAGGTTCTCCAGGTCCAGCTGGTGCTCAAGGTGCTAGAGGTGATGCTGGTGCTAGAGGTGCTAACGGTCCAGCTGGTCCACAAGGTTTTCCAGGTCCAGCTGGTAGACCAGGTTCTCCAGGTGCTAGAGGTGAAACTGGTCCATCTGGTATTAGAGGTGAAACTGGTCCAGCTGGTGCTGCTGGTGCTACTGGTGATCCAGGTGCTCAAGGTCCAGCTGGTGCTGTTGGTGCTCAAGGTGAAAGAGGTGAAGCTGGTAACACTGGTCCACAAGGTCCAGTTGGTAACCCAGGTATTGGTGGTCCACAAGGTAACCAAGGTCCACCAGGTCCAGCTGGTCAAACTGGTCCATCTGGTGCTCCAGGTGCTCCAGGTGAAAGAGGTGATCCAGGTGTTGCTGGTAGAGATGGTGATCAAGGTCCAGTTGGTGCTCCAGGTGCTTCTGGTGAAAAG.

[0116] The nucleotide sequence AS-1, the nucleotide sequence AS-2 and the nucleotide sequence AS-3 were respectively recombined into the plasmid pPIC9K (Thermo v17520) to obtain the recombinant plasmid pPIC9K-1, the recombinant plasmid pPIC9K-2 and the recombinant plasmid pPIC9K-3; the nucleotide sequence AS-1, the nucleotide sequence AS-2 and the nucleotide sequence AS-3 were respectively recombined into the pPICZαA (Thermo v19520) to obtain the recombinant plasmid pPICZαA-1, the recombinant plasmid pPICZαA-2 and the recombinant plasmid pPICZαA-3; the nucleotide sequence AS-1, the nucleotide sequence AS-2 and the nucleotide sequence AS-3 were respectively recombined into the pHIL-S1 (Thermo VT1346) to obtain the recombinant plasmid pHIL-S1-1, the recombinant plasmid pHIL-S1-2 and the recombinant plasmid pHIL-S1-3. The construction of all the above recombinant plasmids was entrusted to Nanjing Ruiguan Biological and was verified to be qualified.

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

[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 allow their OD... 600 =1.3-1.5, take 80 μl of competent cells, add 6 μg of linearized plasmid, and set the electroporation parameters (1.5 kV, 25 μF, 200 Ω). 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 expression and screening:

[0123] 1) Shake flask culture medium formula

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

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

[0126] 2) Respectively pick 9 positive clones of the strains, inoculate in 30 ml of BMGY medium, cultivate at 29°C, 200 rpm for 2 days, sample before induction, centrifuge at 5000 rpm for 5 min, collect the bacteria, wash once with water. Transfer 30 mL of BMMY medium for methanol induction expression, supplement with methanol for induction expression, control the methanol concentration at 0.5-1% / 24h / bottle. Collect the supernatant and precipitate of the shake flask after 3 days of induction, detect by SDS-PAGE electrophoresis, and screen to obtain the expression strains.

[0127] The electrophoretic pictures of the proteins in each culture solution are shown in Figure 1 , wherein 1-9 respectively represent the protein electrophoretic pictures in the supernatant after the genetic engineering bacteria GS115-1, genetic engineering bacteria GS115-2, genetic engineering bacteria GS115-3, genetic engineering bacteria X33-1, genetic engineering bacteria X33-2, genetic engineering bacteria X33-3, genetic engineering bacteria SMD1168-1, genetic engineering bacteria SMD1168-2 and genetic engineering bacteria SMD1168-3.

[0128] 10 L fermenter expression and purification :

[0129] 1) 10L fermenter, the culture medium is BSM medium, which is prepared as follows:

[0130] Fermentation medium: including ammonium phosphate 23.8 / L, potassium phosphate 5.03 g / L, CaSO4·2H2O 0.59 g / L, K2SO49.1 g / L, MgSO4·7H2O 7.45 g / L, glycerol 40 g / L; after high-temperature sterilization of the fermentation medium, wait for the temperature to drop to room temperature, add PTM 12.20 ml / L, and adjust the pH to 5.0 with ammonia water.

[0131] Supplement medium: 50% W / V glycerol, add 12 mL PTM1 trace elements per liter;

[0132] Induction medium: 100% methanol, add 12 mL PTM1 trace elements per liter.

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

[0134] 3) Induction: control the wet weight of the bacteria at about 100-120g / L for induction, first starve for 0.5-1h, then perform methanol induction, interval flow methanol, control the methanol concentration at 0.5-1% for induction, induction for 90h.

[0135] 4) Result determination: after solid-liquid separation, take 5ml of supernatant, incubate with excess NIFF (NTA) filler (Bluex Technology, A4023205), after washing, perform 200mM imidazole elution, collect enough eluate, then use ultrafiltration concentration tube (Thermo, 6kd, 50ml) to desalt and concentrate, control the volume at 1ml, finally use BCA kit to determine the protein concentration.

[0136] Standard curve preparation, standard sample uses laboratory purified target protein, according to the following Table 1 concentration, then use BCA kit (Bi Yun Tian p0011), use enzyme label instrument (Thermo Multiskan FC) absorbance A540 to measure the absorbance, x axis represents standard protein concentration, y axis represents absorbance, formula y = 1.8276x + 0.0308, see Figure 2 for specific standard curve. Respectively determine the absorbance of the eluate, then calculate the protein concentration of the eluate by the formula x = (y-0.0308) / 1.8276, measure three parallel samples for each sample, finally calculate the average value after absorbance test, and finally convert to the concentration of the fermentation tank.

[0137] Table 1 BCA reagent standard curve preparation

[0138]

[0139] Table 2 Expression results of different strains in 10L fermentation tank

[0140]

[0141] The protein expression of different strains is shown in Table 2. Further verification by 10L fermentation tank, the protein expression of the genetically engineered bacteria X33-1 is the best, so the genetically engineered bacteria X33-1 (i.e. Pichia sp.) was preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 18, 2025, the address is No. 1, Beichen West Road, Haidian District, Beijing, China, the Institute of Microbiology of the Chinese Academy of Sciences, the postcode is 100101, the preservation number is CGMCC No. 34894.

[0142] Purification

[0143] 1) After fermentation, solid-liquid separation was carried out, centrifugation was carried out at 4000 rpm for 30 min, and the supernatant was collected.

[0144] 2) Ultrafiltration desalination, using an organic membrane with a molecular weight of 6 kDa for ultrafiltration concentration.

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

[0146] 4) Ultrafiltration desalination, using an organic membrane with a molecular weight of 6 kDa for ultrafiltration desalination.

[0147] 5) Freeze-drying.

[0148] Comparative Example 1. Protein

[0149] Amino acid sequence 2, the amino acid sequence of which is selected from the 35th-248th amino acid sequence in NCBI database collagen

[0150] [Apostichopus japonicus] GenBank: APA22677.1, consisting of 214 amino acids, the amino acid sequence of the protein is specifically shown as SEQ ID NO. 5:

[0151] GLTGPKGPKGEPGQTPVIGSGDLIPGPPGPPGGRGGPGDRGPLGPKGNIGIPGPRGGYGADGVPGLPGPPGPPGPPGPSGQLTAEAQVSQQYKGPVMPAGLGYPQAMMAGEPGQRGPPGQAGLRGPAGLTGAPGEPGEQGPTGPVGLTGPAGSPGRPGSAGKDGANGNPGPAGPSGPPGPAGQNGFPGPSGPKGHRGYSGRAGQKGERGDDGER.

[0152] The above amino acid sequence, such as SEQ ID NO. 5 protein, was codon-optimized by referring to the method of Example 1, and synthesized in a pPICZαA plasmid to obtain a recombinant plasmid pPICZαA-4. Then, the obtained recombinant plasmid pPICZαA-4 was transformed into the host cell Pichia pastoris X33 by referring to the method of Example 2 to obtain a genetically engineered bacterium X33-4. Subsequently, by referring to the method of Example 3, the obtained genetically engineered bacterium X33-4 was expressed in a BMMY culture medium in a shake flask; and the obtained culture supernatant was subjected to protein electrophoresis, and the result is as shown in FIG. 1.Figure 3 As shown in the figure: 1 represents the protein expression result of the shake flask of the genetically engineered bacteria X33-4, and the protein expression amount is very low, which does not meet the requirement of further engineering.

[0153] Comparative Example 2. Protein

[0154] Amino acid sequence 3, the amino acid sequence of which is selected from the 441-680 amino acid sequence in NCBI database collagen

[0155] [Apostichopus japonicus] GenBank: APA22677.1, consisting of 240 amino acids, the amino acid sequence of the protein is specifically shown as SEQ ID NO. 6:

[0156] GPQGPAGDRGQLGAPGSIGPAGAAGAKGETGARGGPGSPGSKGSRGEPGRNGDPGLPGTRGLSGNPGKLGKDGSPGPPGPPGKNGRQGATGATGQRGMPGAQGLTGAPGAQGDRGGDGQVGPPGPAGPQGEVGDRGNPGPAGAPGSPGVAGERGAPGLQGPQGQQGPAGLNGPQGESGAPGDAGPQGETGAPGNPGDRGERGGPGDRGPPGPSGRPGGRGVSGRAGQDGATGAPGPKGEQ.

[0157] The above-mentioned protein with amino acid sequence as SEQ ID NO. 6 is codon-optimized by referring to the method of Example 1, and then synthesized in pPICZαA plasmid to obtain a recombinant plasmid pPICZαA-5. Then, the obtained recombinant plasmid pPICZαA-5 is transformed into the host cell Pichia pastoris X33 by referring to the method of Example 2 to obtain genetically engineered bacteria X33-5. Subsequently, the obtained genetically engineered bacteria X33-5 is expressed by shake flask culture with BMMY medium by referring to the method of Example 3; and the obtained culture supernatant is subjected to protein electrophoresis, and the result is shown in the figure: Figure 3 As shown in the figure: 1 and 2 respectively represent the protein expression results of the shake flask of the genetically engineered bacteria X33-4 and X33-5, and the protein expression amounts of the two genetically engineered bacteria are both too low to meet the requirement of further engineering.

[0158] Example 4. Efficacy test

[0159] 1. Skin repair effect test

[0160] (1) Test principle

[0161] Cell migration refers to the movement of cells after receiving migration signals or sensing the action of certain substances, which is a physiological process of normal growth and development of the body. On human skin, cell migration often occurs in the healing process of wounds, which is similar to the purpose of skin repair efficacy, so the effect of samples on skin repair function can be evaluated to some extent by the migration ability of human keratinocytes. In this test, a human immortalized keratinocyte (HaCaT) scratch model was established, and the repair efficacy of the test sample was evaluated by the effect of the sample on cell migration after the sample was applied.

[0162] (2) Test material

[0163] ① Test system

[0164] The cells used in this test were human immortalized keratinocytes (HaCaT), provided by Zhejiang Meisen Cell Technology Co., Ltd.

[0165] ② Main reagents

[0166] DMEM culture medium (Quanjin, FI101-01), fetal bovine serum (Quanjin, FS301-02), trypsin-EDTA (Quanjin, FG301-01), EGF (MCE, HY-P7109),

[0167] PBS (Biosharp, BL302A).

[0168] ③ Main equipment

[0169] CO2 incubator (Shanghai Lishen, HF90), biological safety cabinet (Suzhou Antai, BSC-1304IIA2), inverted microscope (Motic, AE2000-T), multifunctional enzyme label instrument (TECAN, INFINITE EPLEX).

[0170] ④ Solution preparation

[0171] Sample solution: weigh the test sample, dilute to 0.05%, 0.2%, 0.8% (m / m) with culture medium;

[0172] EGF solution: weigh a certain amount of EGF powder, dissolve in PBS, and dilute to 10 ng / mL with culture medium when used.

[0173] (3) Test method

[0174] ① Sample treatment and grouping: according to the cell viability test results, 0.05%, 0.2%, 0.8% (m / m) sample groups were used in this test, and negative control group and positive control group (10 ng / mL EGF) were set.

[0175] ② Cell seeding: Draw a straight line across the wells 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%.

[0176] 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.

[0177] 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.

[0178] 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 the initial position using the same parameters.

[0179] 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%.

[0180] 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.

[0181] 2. Antioxidant effect

[0182] (1) Test Principle

[0183] 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 single-electron pairing. 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.

[0184] (2) Test materials

[0185] ①Main reagents

[0186] DPPH (Macklin), Vitamin E (Macklin), anhydrous ethanol (National Pharmaceutical Group).

[0187] 2. Main equipment

[0188] Multifunctional enzyme label (TECAN, INFINITE E PLEX), balance (Dolish).

[0189] (3) Test method

[0190] Sample concentration setting

[0191] The sample was diluted with water, mixed uniformly, and then set to the concentration shown in Table 3 for standby.

[0192] Table 3 Sample concentration design table

[0193]

[0194] (4) Determination

[0195] Referring to Table 4, sample tubes (T), sample background tubes (T0), DPPH tubes (C), and solvent background tubes (C0) were set up. For each sample, 3 parallel tubes were set up for each test concentration of the sample tube (T), and 3 parallel tubes were also set up for the DPPH tube (C). In the sample tube (T), 250 μL of sample solution, 500 μL of sample solvent, and 250 μL of DPPH ethanol solution were added. In the sample background (T0), 250 μL of sample solution, 500 μL of sample solvent, and 250 μL of 95% ethanol solution were added. In the DPPH tube (C), 750 μL of sample solvent and 250 μL of DPPH ethanol solution were added, and in the solvent background (C0), 750 μL of sample solvent and 250 μL of 95% ethanol solution were added. Mix well, stand at room temperature for 5 min, and then add each reaction solution to a 96-well plate at 200 μL / well. Measure the absorbance value at 517 nm.

[0196] Table 4 Sample addition setting table

[0197]

[0198]

[0199] (5) Calculation formula

[0200]

[0201] In the formula:

[0202] T - sample absorbance value, i.e. the absorbance value of the solution after the sample and DPPH reaction;

[0203] T0 - sample background absorbance value;

[0204] C- DPPH solution absorbance value without sample addition;

[0205] C0- Solvent background absorbance value.

[0206] (6) DPPH scavenging rate of sample

[0207] According to the DPPH scavenging rate operation procedure, the DPPH scavenging rate of recombinant sea cucumber collagen at various concentrations was detected, as shown in Table 5 below.

[0208] Table 5 DPPH scavenging rate of recombinant sea cucumber collagen at different concentration gradients

[0209]

[0210] According to the test, referring to Table 5, the protein obtained by culturing the genetically engineered bacteria X33-1 using the method of Example 3 above has a DPPH scavenging rate of 31.52%, 37.02%, 47.73%, 50.32%, and 59.28% at a concentration of 1.25-20 wt%, respectively. The test evaluates the antioxidant effect of the sample by detecting the ability of the sample to scavenge DPPH free radicals, so the results show that the sample has a certain antioxidant effect.

[0211] 47.73%, 50.32%, and 59.28%, respectively. The test evaluates the antioxidant effect of the sample by detecting the ability of the sample to scavenge DPPH free radicals, so the results show that the sample has a certain antioxidant effect.

[0212] Example 5. Emulsion

[0213] The protein obtained by culturing the genetically engineered bacteria X33-1 using the method of Example 3 above was used to prepare an emulsion, and the formula of the emulsion is shown in Table 6.

[0214] Table 6 Components and contents of emulsion

[0215]

[0216]

[0217] Example 6. Repairing dressing

[0218] The protein obtained by culturing the genetically engineered bacteria X33-1 using the method of Example 3 above was used to prepare a repairing dressing, and the formula of the repairing dressing is shown in Table 7.

[0219] Table 7 Components and contents of repairing dressing

[0220]

[0221]

[0222] Example 7. Cream dressing

[0223] 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.

[0224] Table 8. Components and Content of Each Component in Repair Dressing

[0225]

[0226] 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 implementation 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 this disclosure to the disclosed forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical applications of this disclosure.

Claims

1. A protein with repair function, having an amino acid sequence as shown in SEQ ID NO.

1.

2. The protein according to claim 1, wherein the nucleotide sequence encoding the protein is shown in any one of SEQ ID NO. 2 to 4.

3. A method for preparing the protein with repair efficacy as described in claim 1 or 2, 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.

4. The preparation method according to claim 3, 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.

5. The preparation method according to claim 3 or 4, 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.

6. The preparation method according to claim 5, 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 is 0.5-1 wt% during methanol induction; 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; Preferably, the solid-liquid separation method includes any one or more of centrifugation, microfiltration, vacuum filtration, and ultrafiltration; Preferably, the chromatography includes any one or more of ion chromatography and hydrophobic chromatography.

7. A recombinant Pichia pastoris expressing the amino acid sequence shown in SEQ ID NO. 1; and / or, It contains nucleotide sequences as shown in any one of SEQ ID NO. 2 to 4.

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

9. Use of the protein according to claim 1 or 2 for promoting skin repair.

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

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