Polypeptide and use thereof in cosmetics

Abstract

The present invention relates to multiple polypeptides with high safety, cell repair capacity, and anti-inflammatory, hydrating and moisturizing effects, and the use thereof in cosmetics, wherein the amino acid sequences of the polypeptides are as set forth in SEQ ID NO: 1-SEQ ID NO: 6. The present invention also relates to a polynucleotide encoding the polypeptides, wherein the sequence of the polynucleotide is as set forth in SEQ ID NO: 7-SEQ ID NO: 12, and the sequence of the polynucleotide can be used for the detection of Deinococcus weizhi. In addition, further provided is a topical drug or a cosmetic, which contains any one of the polypeptides or a combination thereof as an active ingredient.

Description

Peptides and their applications in cosmetics

[0001] Priority information

[0002] This application claims priority to Chinese patent application No. 202410253984.8 filed with the Patent Office of China on March 6, 2024, entitled “Polypeptides and Their Application in Cosmetics,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a variety of polypeptides with high safety, cell repair ability, anti-inflammatory and hydrating and moisturizing effects, and uses thereof in cosmetics. Background Art

[0004] Peptides are a class of compounds composed of amino acids linked by peptide bonds and are ubiquitous chemically active substances in living organisms. According to the commonly accepted classification system in the international pharmaceutical community, drugs with more than 100 amino acid molecules are classified as protein drugs, while those with fewer than 100 are classified as polypeptide drugs. Currently, tens of thousands of peptides have been discovered in living organisms, and most possess physiological activities involved in various fields, including hormones, nerves, cell growth, and reproduction.

[0005] Peptides are active molecules composed of a series of amino acids linked by specific sequences. They are widely found in plants and animals. According to the international pharmaceutical classification, drugs with more than 100 amino acid molecules are classified as protein drugs, while those with less than 100 are classified as polypeptide drugs. Currently, tens of thousands of polypeptides have been discovered in organisms, and most have physiological activities involved in various fields such as hormones, nerves, cell growth, and reproduction. Compared with chemical drugs, polypeptide drugs are more effective, safer, and more tolerable. Since the 1970s, small molecule peptides with various biological functions have been discovered to easily cross the skin barrier and reach the matrix, which can improve a range of skin problems, ushering in an era of pharmaceutical polypeptide raw materials. Currently, among the dozens of pharmaceutical peptide raw materials used in China, there are eight peptides of natural origin. The discovery of natural polypeptides has provided more options for raw materials in pharmaceutical peptides. Summary of the Invention

[0006] The first aspect of the present invention provides a polypeptide. According to an embodiment of the present invention, the polypeptide sequence has at least one of the amino acids shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0007] The polypeptides provided by the present invention have high safety, strong cell repair capabilities, anti-inflammatory, scar-lightening, and hydrating properties. The polypeptides of the present invention are derived from Deinococcus microti. Through further genomic research, the inventors analyzed the differences between the genomes of Deinococcus microti and other Deinococcus species, discovered a series of exocrine polypeptides unique to Deinococcus microti and isolated several with repair and anti-inflammatory properties. Based on the amino acid sequences of the isolated polypeptides, those skilled in the art can more quickly obtain the polypeptides of the present invention through chemical synthesis.

[0008] In a specific embodiment of the present invention, peptide 1 has the amino acid sequence shown in SEQ ID NO: 1: ASPTVAVHFPSGQTVKVPVPKSGDVALAQARLMRYSRLTVA (SEQ ID NO: 1);

[0009] Peptide 2 has the amino acid sequence shown in SEQ ID NO: 2: LDSLIPLSPAPLDNCR (SEQ ID NO: 2);

[0010] Peptide 3 has the amino acid sequence shown in SEQ ID NO: 3: CTGAKEGT (SEQ ID NO: 3);

[0011] Peptide 4 has the amino acid sequence shown in SEQ ID NO: 4: APSD (SEQ ID NO: 4);

[0012] Peptide 5 has the amino acid sequence shown in SEQ ID NO: 5: SPVQFSGLRPTC (SEQ ID NO: 5);

[0013] Peptide 6 has the amino acid sequence shown in SEQ ID NO: 6: FIGSSGNFLTSSFCTGVAKCAKTRFLS (SEQ ID NO: 6).

[0014] The term "peptide" used in this specification may refer to a linear molecule formed by amino acid residues bound to each other through peptide bonding. The peptide can be prepared according to chemical synthesis methods known in the art, especially according to solid phase synthesis technology.

[0015] The second aspect of the present invention provides an isolated polynucleotide encoding the polypeptide of the first aspect.

[0016] According to a specific embodiment of the present invention, the polynucleotide has at least one of the nucleotide sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0017] The present invention provides a polynucleotide encoding the peptide 1. Specifically, the polynucleotide sequence is as shown in SEQ ID NO: 7:

[0018] The present invention provides a polynucleotide encoding the peptide 2. Specifically, the polynucleotide sequence is as shown in SEQ ID NO: 8:

[0019] The present invention provides a polynucleotide encoding the peptide 3. Specifically, the polynucleotide sequence is as shown in SEQ ID NO: 9:

[0020] The present invention provides a polynucleotide encoding the peptide 4. Specifically, the polynucleotide sequence is as shown in SEQ ID NO: 10:

[0021] The present invention provides a polynucleotide encoding the peptide 5. Specifically, the polynucleotide sequence is as shown in SEQ ID NO: 11:

[0022] The present invention provides a polynucleotide encoding the peptide 6. Specifically, the polynucleotide sequence is as shown in SEQ ID NO: 12:

[0023] It should be noted that, for the nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other strand complementary thereto is also disclosed. In addition, the nucleic acid sequences in this application include DNA forms or RNA forms, and disclosure of one of them means that the other is also disclosed. Those skilled in the art can easily synthesize the polypeptides of the present invention using the nucleic acid sequences or amino acid sequences provided by the present invention.

[0024] The third aspect of the present invention is to provide a plurality of expression vectors, wherein the expression vectors comprise the polynucleotides as described above.

[0025] According to a specific embodiment of the present invention, the expression vector comprises at least one of the polynucleotides shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0026] The expression vector 1 comprises the polynucleotide shown in SEQ ID NO: 7;

[0027] The expression vector 2 comprises the polynucleotide shown in SEQ ID NO: 8;

[0028] The expression vector 3 comprises the polynucleotide described in SEQ ID NO: 9;

[0029] The expression vector 4 comprises the polynucleotide shown in SEQ ID NO: 10;

[0030] The expression vector 5 comprises the polynucleotide shown in SEQ ID NO: 11;

[0031] The expression vector 6 comprises the polynucleotide shown in SEQ ID NO: 12;

[0032] The expression vector 7 contains the polynucleotides shown in SEQ ID NO:7 and SEQ ID NO:8, the expression vector 8 contains the polynucleotides shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:12, and the expression vector 9 contains the polynucleotides shown in SEQ ID NO:8 and SEQ ID NO:12.

[0033] According to an embodiment of the present invention, the expression vector may include optional control sequences that are operably linked to the nucleic acid molecule. The control sequences are one or more control sequences that direct the expression of the nucleic acid molecule in a host. The expression vectors proposed in the embodiments of the present invention can efficiently express the polypeptide in large quantities in suitable host cells.

[0034] The fourth aspect of the present invention is to provide a recombinant cell carrying the above-mentioned polynucleotide, the above-mentioned expression vector or capable of expressing the above-mentioned polypeptide.

[0035] According to a specific embodiment of the present invention, the recombinant cell is obtained by introducing the aforementioned expression vector into a host cell.

[0036] It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptococcus, or Proteus mirabilis, among others. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as fall armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0037] The host cell 1 is transformed and / or transfected with the expression vector 1;

[0038] The host cell 2 is transformed and / or transfected with the expression vector 2;

[0039] The host cell 3 is transformed and / or transfected with the expression vector 3;

[0040] The host cell 4 is transformed and / or transfected with the expression vector 4;

[0041] The host cell 5 is transformed and / or transfected with the expression vector 5;

[0042] The host cell 6 is transformed and / or transfected with the expression vector 6;

[0043] The host cell 7 is transformed and / or transfected with the expression vector 7, the host cell 8 is transformed and / or transfected with the expression vector 8, and the host cell 9 is transformed and / or transfected with the expression vector 9.

[0044] The fifth aspect of the present invention provides a composition comprising at least one of the aforementioned polypeptides, polynucleotides, expression vectors, recombinant cells, and compositions.

[0045] The sixth aspect of the present invention is to provide a drug for external use, which comprises at least one of the aforementioned polypeptides, polynucleotides, expression vectors, recombinant cells, and compositions.

[0046] According to a specific embodiment of the present invention, the drug comprises any one of the polypeptides of SEQ ID NOs: 1-6 or a combination thereof as an active ingredient.

[0047] According to a specific embodiment of the present invention, the external medicine includes dosage forms such as ointment, pill, water, wine, powder, and medicine thread (medicine tablet), which can be used directly. The usage includes plaster, coating, application, mixing, fumigation, washing, soaking, bathing, eye drops, ear irrigation, nasal drops, etc.

[0048] According to a specific embodiment of the present invention, the external drug dosage forms include solution type, suspension type, emulsion type; ointment, which can also be divided into ointment, cream, gel; tincture, liniment, spirit, powder, oil, paste, plaster, film coating, aerosol, etc.

[0049] The seventh aspect of the present invention is to provide a cosmetic comprising at least one of the aforementioned polypeptide, polynucleotide, expression vector, recombinant cell, and composition.

[0050] According to a specific embodiment of the present invention, the cosmetic comprises any one of the polypeptides of SEQ ID NOs: 1-6 or a combination thereof as an active ingredient.

[0051] According to the "Regulations on the Management of Cosmetics Labels", cosmetics refer to products that are applied to the human body (skin, hair, nails, lips, teeth, etc.) by smearing, spraying, drinking or other similar methods to achieve the purpose of cleaning, maintenance, beautification, modification and change of appearance, or to correct human odor and maintain good condition.

[0052] According to a specific embodiment of the present invention, the cosmetics include cleaning products, skin care products, oral products, etc.

[0053] An eighth aspect of the present invention provides the use of the aforementioned polypeptide, polynucleotide, expression vector, recombinant cell, and composition in the preparation of cosmetics.

[0054] According to a specific embodiment of the present invention, the cosmetic has at least one of the effects of anti-inflammation, scar reduction, and moisturizing.

[0055] According to a specific embodiment of the present invention, the cosmetic has at least one of the functions of promoting skin wound healing and / or scar repair.

[0056] The ninth aspect of the present invention provides the use of the aforementioned polypeptide, polynucleotide, expression vector, recombinant cell, and composition in the preparation of a drug.

[0057] According to a specific embodiment of the present invention, the drug has at least one of the effects of anti-inflammation, accelerating wound healing of the skin surface, and reducing scar formation.

[0058] According to a specific embodiment of the present invention, the drug has at least one of the effects of promoting skin wound healing and / or scar repair.

[0059] The tenth aspect of the present invention provides the use of the aforementioned polynucleotide in the preparation of a detection kit for identifying Deinococcus microti.

[0060] The eleventh aspect of the present invention provides a kit for detecting Deinococcus microti, comprising the polynucleotide described in the second aspect.

[0061] The twelfth aspect of the present invention provides use of the polypeptide described in the first aspect and / or the polynucleotide described in the second aspect in detecting Deinococcus microti.

[0062] A thirteenth aspect of the present invention provides a method for identifying Deinococcus microti, comprising:

[0063] (1) designing a primer pair that is reverse complementary to the sequence of the polynucleotide described in the second aspect;

[0064] (2) using the primers to amplify the sample to be tested; or

[0065] (3) contacting the polypeptide described in the first aspect with a sample to be tested;

[0066] Wherein, the sample to be tested is a sample containing Deinococcus microti.

[0067] A fourteenth aspect of the present invention provides a method for identifying Deinococcus microti, comprising:

[0068] (1) designing a primer pair that is reverse complementary to the sequence of the polynucleotide described in the second aspect;

[0069] (2) using the primers to amplify the sample to be tested,

[0070] Wherein, the sample to be tested from which the nucleic acid fragment can be amplified is a sample containing Deinococcus microti.

[0071] According to an embodiment of the present invention, the method for identifying Deinococcus microti in the thirteenth and fourteenth aspects may further include at least one of the following additional technical features:

[0072] According to a specific embodiment of the present invention, the primer pair has at least one of the following nucleotide sequences:

[0073] (a) the nucleotide sequences described in SEQ ID NO: 13 and SEQ ID NO: 14;

[0074] (b) the nucleotide sequences described in SEQ ID NO: 15 and SEQ ID NO: 16;

[0075] (c) the nucleotide sequences described in SEQ ID NO: 17 and SEQ ID NO: 18;

[0076] (d) the nucleotide sequences described in SEQ ID NO: 19 and SEQ ID NO: 20;

[0077] (e) the nucleotide sequences described in SEQ ID NO: 21 and SEQ ID NO: 22;

[0078] (f) The nucleotide sequences described in SEQ ID NO: 23 and SEQ ID NO: 24.

[0079] According to a specific embodiment of the present invention, peptide 1-peptide 6 are secretory polypeptides unique to Microdeinococcus, and the nucleotide sequence encoding peptide 1-peptide 6 is also a nucleotide sequence unique to Microdeinococcus. By detecting the presence or absence of any one of the unique nucleic acid sequences, Microdeinococcus can be identified among numerous bacterial populations.

[0080] According to a specific embodiment of the present invention, the cosmetic and / or medicine has at least one of the effects of anti-inflammation, accelerating wound healing of the skin surface, and reducing scar formation.

[0081] According to a specific embodiment of the present invention, the cosmetic and / or medicine has at least one of the functions of promoting skin wound healing and / or scar repair.

[0082] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG1 shows a graph showing the activity of the polypeptide in Example 4 of the present invention in promoting scratch repair in HaCaT cells;

[0084] FIG2 shows the tail area of ​​zebrafish in the peptide moisturizing and hydrating efficacy test in Example 6 of the present invention;

[0085] FIG3 shows the moisturizing and hydrating rate (%) of the polypeptide in Example 6 of the present invention;

[0086] Figure 4 shows the nucleic acid electrophoresis diagram of the PCR products of the polypeptide nucleotide positions of Deinococcus urumqiensis R12, Deinococcus radiodurans DSM20539, and Deinococcus microcaulis VB142 in Example 7 of the present invention. DETAILED DESCRIPTION

[0087] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0088] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0089] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0090] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0091] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0092] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0093] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 10:106); the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 10:116; the similarity search method of Pearson et al. (1990) Proc. Natl. Acad. Sci. 10:117; the similarity search method of Pearson et al. (1990 ... .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0094] As used herein, the term "at least 70% identity" refers to a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to its corresponding reference sequence, including amino acid sequences, nucleic acid sequences, fusion sequences of DNA and RNA, or fusion sequences of amino acids and nucleic acids.

[0095] As used herein, the term "nucleotide" refers to ribonucleotides, deoxynucleotides or modified forms of either type of nucleotide, and combinations thereof.

[0096] As used herein, the term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced.

[0097] As used herein, the term "transformed" or "transfected" refers to the introduction of a nucleic acid (eg, a vector) into a cell by various techniques known in the art.

[0098] As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0099] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is encompassed.

[0100] In this document, the term "pharmaceutically acceptable excipients" may include sugars, including monosaccharides or polysaccharides, such as lactose, sucrose, mannitol and sorbitol; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil; nonionic surfactants, cationic surfactants, anionic surfactants; ethylene glycol polymers; fatty alcohols; and cereal hydrolyzed solids and other non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants and the like excipients commonly used in pharmaceutical preparations.

[0101] peptides

[0102] The present invention provides a polypeptide, the polypeptide sequence having at least one of the amino acids shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0103] The polypeptides provided herein have high safety, strong cell repair capabilities, anti-inflammatory, and hydrating properties. The polypeptides of the present invention are derived from Deinococcus microti. Through further genomic research, the inventors analyzed the differences between the genomes of Deinococcus microti and other Deinococcus species, discovered a series of exocrine polypeptides unique to Deinococcus microti and isolated several with repair and anti-inflammatory properties. Based on the amino acid sequences of the isolated polypeptides, those skilled in the art can more rapidly synthesize the polypeptides of the present invention through chemical synthesis.

[0104] According to specific embodiments of the present invention, the polypeptides provided herein can be directly chemically synthesized, isolated from a secretory polypeptide of Deinococcus weizhi, or transferred into other host cells for secretory expression. The present invention provides the amino acid sequences of the aforementioned polypeptides. Those skilled in the art can obtain the polypeptides described herein using existing techniques, and any method known in the art for obtaining polypeptides is within the scope of protection of the present invention. The synthesized polypeptides can be directly stored in powdered form for ease of storage and transportation.

[0105] According to specific embodiments of the present invention, the polypeptide of the present invention can be widely used in applications such as medicine, cosmetics, and food.

[0106] In a specific embodiment of the present invention, the polypeptide sequence has at least one of the amino acids shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0107] According to a specific embodiment of the present invention, the above-mentioned polypeptides can function alone, for example, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 alone; or they can function in any combination, for example, a combination of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, a combination of SEQ ID NO: 3, SEQ ID NO: 4, a combination of SEQ ID NO: 1, SEQ ID NO: 4, etc.

[0108] Nucleic acid molecules, recombinant vectors, recombinant cells, compositions, medicines, cosmetics

[0109] In the process of preparing or obtaining these polypeptides, nucleic acid molecules expressing these polypeptides can be connected to different vectors and then expressed in different cells to obtain the corresponding polypeptides.

[0110] Therefore, the present invention also provides an isolated nucleic acid molecule (polynucleotide) encoding the above polypeptide, as well as a recombinant vector and a transformant containing the nucleic acid molecule. The nucleic acid molecule encodes the above polypeptide.

[0111] In some embodiments, the nucleic acid has at least one of the nucleotide sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0112] In some specific embodiments, the nucleic acid has at least one of the nucleotide sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0113] In the present invention, the nucleic acid is preferably an expression cassette obtained by genetic engineering means.

[0114] In some preferred embodiments, the nucleic acid molecule is species-optimized and is more easily expressed in mammalian cells.

[0115] The present invention also provides an expression vector comprising the isolated nucleic acid molecule described above. When the isolated polynucleotide is linked to the vector, the polynucleotide may be directly or indirectly linked to control elements on the vector, as long as these control elements are capable of controlling translation and expression of the polynucleotide. These control elements may be derived directly from the vector itself or exogenously, i.e., not derived from the vector itself. It is sufficient that the polynucleotide and the control elements are operably linked.

[0116] The recombinant vector may refer to a cloning vector or an expression vector, and can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). For example, commonly used plasmids include pSeTag2, PEE14, pMH3, and the like.

[0117] The present invention also provides a recombinant cell comprising the expression vector. The expression vector can be introduced into mammalian cells to construct recombinant cells, which can then be used to express the polypeptide provided by the present invention. The corresponding polypeptide can be obtained by culturing the recombinant cells. The host cell of the present invention can be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptococcus, or Proteus mirabilis, etc. The eukaryotic host cell can be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, or mammalian cells such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the host cell of the present invention is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0118] The composition provided by the present invention contains at least one of the polypeptide, polynucleotide, expression vector, recombinant cell, and composition as described above.

[0119] Those skilled in the art will appreciate that the composition includes food compositions, pharmaceutical compositions, and the like.

[0120] According to an embodiment of the present invention, the above-mentioned composition further comprises food or a pharmaceutically acceptable carrier. In some embodiments, the composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of the present invention. For example, the ingredients contained in the composition can be applied to the subject as a whole, or separately applied to the subject. When the ingredients contained in the composition are applied to the subject separately, each ingredient can be applied to the subject simultaneously or sequentially.

[0121] According to an embodiment of the present invention, the present invention provides a medicine or cosmetic containing at least one of the amino acids shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Preferably, the cosmetic of the present application has at least one of the effects of anti-inflammatory, scar lightening, and moisturizing, and the medicine has the effects of anti-inflammatory, accelerating wound healing of the skin surface, reducing scar formation, and scar repair. It should be noted that the medicine or cosmetic of the present application can be various preparations containing the above-mentioned polypeptide alone, or can be used in combination with other active ingredients, as long as they do not affect the activity of each other; furthermore, the best situation for the medicine or cosmetic is that the active ingredients can complement each other or have a promoting effect. For example, the above-mentioned polypeptide can be combined with other probiotics to achieve better or more active functions. The specific effect can be determined according to other active ingredients and components in the medicine or cosmetic, and is not limited here. Optionally, the medicine or cosmetic further includes a cosmetically or pharmaceutically acceptable carrier or excipient, or a pharmaceutically acceptable carrier or excipient. Medicines or skin care products can be in any form such as powders, ointments, patches, emulsions, etc.

[0122] The present invention also provides a use of the above-mentioned polypeptide sequence and nucleotide sequence for identifying Deinococcus microti.

[0123] Sequence analysis: Alignments were performed using the ClustalW sequence alignment method with default parameters. The preferred parameter settings used were: for pairwise alignments: Gap open penalty: 10; Gap extension penalty: 0.1, and for multiple alignments, a gap open penalty of 10 and a gap extension penalty of 0.2. The protein weight matrix was set to Identity. Residue specificity and hydrophobicity penalties were both "on," gap spacing was 4, and terminal gap spacing was "off." No negative matrices were used, and finally, the Delay Divergent Cut-off was set to 30%.

[0124] The present method determines sequence similarity based on sequence identity. In bacterial identification, according to widely accepted guidelines, if the 16S rRNA sequence similarity between two bacteria is less than 97%, it generally indicates that they likely belong to different species. Conversely, if the similarity is greater than 97%, it indicates that the species are taxonomically closer. This threshold, based on extensive research comparing bacterial 16S rRNA sequences, reflects small but significant genetic differences between species.

[0125] For peptide sequence identification, the criteria for determining similarity vary. In the present invention, two peptide sequences are considered similar if their identity reaches or exceeds 80%. This criterion reflects that at the protein level, structural and functional similarities can still be maintained even with a certain degree of sequence divergence.

[0126] Sequence identity: A quantitative measure of the degree of homology between two amino acid sequences or two nucleotide sequences of equal length. If the sequences to be compared are not of equal length, they must be aligned to give the best possible match, allowing for the insertion of gaps or (optionally) truncation at the ends of the polypeptide or nucleotide sequence. Sequence identity can be calculated using where Ndif is the total number of residues that are not identical in the two sequences after alignment, and Nref is the number of residues in one sequence. Thus, the DNA sequence AGTCAGTC and the sequence AATCAATC (Ndif=2 and Nref=8) have 75% sequence identity. Gaps are calculated as the inconsistency of specific residues, i.e., the DNA sequence AGTGTC and the DNA sequence AGTCAGTC (Ndif=2 and Nrep=8) have 75% sequence identity. For all embodiments of the present invention relating to amino acid sequences, the percentage of sequence identity between one or more sequences can also be based on an alignment performed using clustalW software (http: / / www.ebi.ac.uk / clustalW / index.html) using default settings.

[0127] After a full species comparison based on bioinformatics analysis, the amino acid sequence encoded by polypeptides 1-6 is a sequence unique to Microdeinococcus. Those skilled in the art can use the amino acid sequences of polypeptides 1-6 or the corresponding nucleic acid sequences provided by the present invention to detect whether the bacterial population contains Microdeinococcus and identify the species of Microdeinococcus. This is more convenient and faster than whole genome sequencing and more accurate than 16S sequencing.

[0128] In some embodiments, the present invention provides a method for identifying Deinococcus microti, comprising:

[0129] (1) designing a primer pair that is reverse complementary to the sequence of the aforementioned polynucleotide;

[0130] (2) using the primers to amplify the sample to be tested; or

[0131] (3) contacting the polypeptide described above with the sample to be tested;

[0132] Wherein, the sample to be tested is a sample containing Deinococcus microti.

[0133] In some embodiments, the present invention provides a method for identifying Deinococcus microti, comprising:

[0134] (1) designing a primer pair that is reverse complementary to the sequence of the aforementioned polynucleotide;

[0135] (2) using the primers to amplify the sample to be tested,

[0136] Wherein, the sample to be tested from which the nucleic acid fragment can be amplified is a sample containing Deinococcus microti.

[0137] In some embodiments, the above-mentioned method for identifying Deinococcus microti may further include at least one of the following additional technical features:

[0138] In some specific embodiments, the primer pair has at least one of the following nucleotide sequences:

[0139] (a) the nucleotide sequences described in SEQ ID NO: 13 and SEQ ID NO: 14;

[0140] (b) the nucleotide sequences described in SEQ ID NO: 15 and SEQ ID NO: 16;

[0141] (c) the nucleotide sequences described in SEQ ID NO: 17 and SEQ ID NO: 18;

[0142] (d) the nucleotide sequences described in SEQ ID NO: 19 and SEQ ID NO: 20;

[0143] (e) the nucleotide sequences described in SEQ ID NO: 21 and SEQ ID NO: 22;

[0144] (f) The nucleotide sequences described in SEQ ID NO: 23 and SEQ ID NO: 24.

[0145] In some specific embodiments, peptide 1-peptide 6 are secretory polypeptides unique to Microdeinococcus, and the nucleotide sequence encoding peptide 1-peptide 6 is also a nucleotide sequence unique to Microdeinococcus. By detecting the presence or absence of any one of the unique nucleic acid sequences, Microdeinococcus can be identified among many bacterial populations.

[0146] In some specific embodiments, the cosmetic and / or medicine has at least one of the effects of anti-inflammation, accelerating wound healing of the skin surface, and reducing scar formation.

[0147] In some specific embodiments, the cosmetic and / or medicine has at least one of the functions of promoting skin wound healing and / or scar repair.

[0148] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0149] Example 1: Isolation and identification of VB142

[0150] 1. Separation of VB142

[0151] The strain VB142 with anti-ultraviolet properties was isolated and screened from the facial skin of adult women. The isolation method is as follows: using 20% ​​glycerol as the sampling liquid, collect samples from the facial skin of adult women and add them to the sampling liquid. Take 100uL and spread it on solid culture medium, culture at 30℃ for 72h, select an orange-red strain, and obtain VB142 after repeated purification.

[0152] 2. Identification of VB142

[0153] (1) Morphological characteristics: When VB142 was cultured in TGY solid medium at 30°C for 72 h, the bacteria were spherical, and the colonies were round, small, convex, smooth, opaque, and orange-red;

[0154] (2) Physiological and biochemical characteristics: The following physiological and biochemical characteristics of VB142 were determined according to the methods of the Manual of Identification of Common Bacteria Systems.

[0155] VB142 is a Gram-positive bacterium and catalase-positive. Its utilization of different carbon sources is shown in Table 1.

[0156] Table 1. Carbon source utilization results of VB142 Note: “+” indicates that the carbon source can be utilized, and “-” indicates that the carbon source cannot be utilized.

[0157] (3) 16S rRNA gene

[0158] The 16S rRNA gene sequence of VB142 is shown in SEQ ID NO: 25. The similarity comparison results show that the most similar 16S rRNA gene sequence to this strain is Deinococcus wulumuqiensis R12 T ), with a sequence identity of 94.28%.

[0159] (4) G+C mol% value

[0160] The G+C mol% content of VB142 is 63.99%.

[0161] (5) Whole genome ANI value

[0162] The whole genome sequence analysis showed that VB142 was similar to Deinococcus wulumuqiensis R12 T ) The ANI value of the comparison was 85.63%.

[0163] Based on morphological, physiological and biochemical characteristics, 16S rRNA gene, and whole-genome sequence analysis, VB142 was designated a new species of the genus Deinococcus, Deinococcus weizhi. It was deposited with the China Center for Type Culture Collection (CCTCC), Wuhan University, under accession number CCTCC M 2024185, on January 23, 2024, or with the Korea Culture Collection (KCTC), under accession number 15470BP, on June 19, 2023. The deposited name is Deinococcus sp.

[0164] The inventors conducted homologous gene analysis on strain VB142 and other Deinococci to determine the differences in the genomes of Microdeinococcus and other Deinococcus species. Specifically, the proteome sequences of each strain were retrieved and downloaded from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) species library, and pan-genome analysis was performed using Orthofinder (https: / / github.com / davidemms / OrthoFinder). A series of exocrine polypeptides unique to Microdeinococcus were discovered, as shown in Table 2. Peptides 1 to 6 are exocrine polypeptides unique to Microdeinococcus.

[0165] Table 2

[0166] The nucleotide sequences of peptides 1 to 6 encoding the secreted polypeptides in Table 2 also only appear in Deinococcus microphylla and are specific to Deinococcus microphylla. Their locations in the genome of Deinococcus microphylla VB142 are shown in Table 3.

[0167] Table 3

[0168] Example 2: Synthetic polypeptide

[0169] Peptides 1 to 6 provided by the present invention can all be prepared using solid phase synthesis technology, including the following steps:

[0170] a) Reactor treatment:

[0171] Add 2 / 3 volume of dichloromethane (DCM, Aladdin) to the reactor and soak for 3 h;

[0172] b) Resin swelling:

[0173] 1.0 g of Wang resin (Sigma-Aldrich) was weighed and added to the reaction column, and 20 mL of N,N-dimethylformamide (DMF, Aladdin) was added and soaked for 30 min to fully swell the resin;

[0174] c) Attachment of the first amino acid:

[0175] A reaction solution containing 8 mmol of Fmoc-modified amino acids (Sigma-Aldrich, peptide 1 is Fmoc-Ala-OH, peptide 2 is Fmoc-Leu-OH, peptide 3 is Fmoc-Cys(tBu)-OH, peptide 4 is Fmoc-Ala-OH, peptide 5 is Fmoc-Leu-OH, and peptide 6 is Fmoc-Phe-OH), 0.9 mmol of 4-dimethylaminopyridine (DMAP, Aladdin), and 4.5 mmol of N,N'-diisopropylcarbodiimide (DIC, Aladdin) was prepared, and nitrogen was introduced and the reaction was carried out for 2.5 hours.

[0176] d) Washing:

[0177] After the reaction was completed, the reaction solution was blown away and washed twice with 10 mL of DCM, isopropanol and DMF respectively.

[0178] e) Blocking of residual reactive groups:

[0179] 0.6 mL of benzoyl chloride (Aladdin) and 0.45 mL of pyridine (Aladdin) were dissolved in 10 mL of DCM, added to the resin, reacted for 1 hour, and then washed twice with 10 mL of DCM, isopropanol, and DMF respectively.

[0180] f) Removal of Fmoc protecting group:

[0181] Add 15 mL of a 20% piperidine solution in DMF to a reaction vessel and allow to react for 5 minutes under nitrogen. After draining, add another 20 mL of a 20% piperidine solution in DMF and allow to react for 20 minutes under nitrogen. After draining, wash twice with 10 mL of DCM, 10 mL of isopropanol, and then 10 mL of DMF. A small amount of resin was tested for removal using the ninhydrin (Sigma-Aldrich) method. If the resin appears black or purple-black, complete removal is indicated.

[0182] g) Amino acid condensation:

[0183] According to the peptide sequence, the second amino acid reaction solution was prepared containing 2 mmol of Fmoc-modified amino acids (peptide 1 was Fmoc-Ser-OH, peptide 2 was Fmoc-Asp(OtBu)-OH, peptide 3 was Fmoc-Thr-OH, peptide 4 was Fmoc-Pro-OH, peptide 5 was Fmoc-Pro-OH, and peptide 6 was Fmoc-Ile-OH), 0.8 g of benzotriazole tetramethyltetrafluoroboric acid (TBTU, Aladdin) in 10 mL of DMF solution, and then 1 mL of 0.135 g / mL p-hydroxybenzonitrile (HoBt, Aladdin) and 0.4 mL of N,N-diisopropylethylamine (DIEA, Aladdin) were added, added to the reactor, and reacted under nitrogen for 2 hours.

[0184] h) Repeat d) → g) according to the amino acid sequence until the last amino acid is synthesized.

[0185] i) Cleavage of peptides:

[0186] A cutting reagent containing 82.5% trichloroacetic acid, 5% thioanisole, 5% water, 5% phenol, and 2.5% ethanedithiol was added and reacted at room temperature for 4 hours. After the reaction was completed, it was fully dried under nitrogen conditions to obtain peptide 1-peptide 6 powder.

[0187] Example 3: Cell proliferation assay

[0188] The safety of the peptide provided by the present invention was investigated using a cell proliferation assay. The solution used in this example was prepared as follows:

[0189] Complete DMEM medium was prepared as follows: 89% Gibco high-glucose DMEM medium (Gibco), 10% fetal bovine serum (Gibco), 1% 100× penicillin-streptomycin double antibody (Sigma-Aldrich);

[0190] Peptide solution of the experimental group: Peptides 1 to 6 prepared in Example 2 were dissolved in complete DMEM medium respectively, with a stock solution concentration of 5 mg / mL, stored at 4° C., and diluted with complete DMEM medium before use.

[0191] The experimental process of this example is as follows: HaCat cells growing in the logarithmic phase were plated on a 96-well plate, and 200 μL of 1×10 4 Cells were cultured using complete DMEM medium. After 24 hours of cell attachment, the medium was discarded and replaced with 100 μL of DMEM complete medium containing peptide 1-peptide 6, with peptide 1-peptide 6 concentrations of 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, and 5 mg / mL, with 3 replicates for each concentration. A blank group containing a DMEM complete medium solution without peptide 1-peptide 6 was also set up. After 24 hours, 100 μL of Cell 1-peptide 6 was added to each well. Luminescent Cell Viability Assay kit (Promega, G7573) was used for detection. The cells were incubated at 37°C for 10 minutes, and the luminescence intensity was measured on a microplate reader to reflect the number of cells.

[0192] The test results are shown in Table 4:

[0193] Table 4: Relative cell numbers in each group of cell proliferation assay

[0194] The results showed that after peptides 1 to 6 were co-incubated with HaCat cells at a concentration below 5 mg / mL for 24 hours, their cell numbers had no significant inhibitory or promoting effects compared with the blank control, indicating that the peptides provided by the present invention are non-cytotoxic and highly safe.

[0195] Example 4: HaCat cell scratch repair function test

[0196] In order to observe the cell repair ability of the peptide provided by the present invention, the scratch repair function test was performed using HaCat cells. The solution used in this example was prepared as follows:

[0197] Complete DMEM medium: 89% high-glucose DMEM medium (Gibco), 10% fetal bovine serum (Gibco), 1% 100× penicillin-streptomycin double antibody (Sigma-Aldrich);

[0198] Peptide solution for the experimental group: The peptide was dissolved in complete DMEM medium at a concentration of 2 mg / mL and stored at 4°C.

[0199] The experimental process of this example is as follows: a scratch wound healing 2-well insert (ibidi) was attached to the center of a 12-well plate, and the concentration of the cell suspension was adjusted to 3×10 570 μL of cell suspension was added to each well of the wound healing two-well insert. After adding the cell suspension, avoid shaking the well plate and incubate at 37°C and 5% CO2 for at least 24 hours. After 24 hours, the cell density was examined under a microscope. After obtaining a confluent cell layer in the two-well insert, the culture insert was gently removed with sterile forceps and the cell layer was washed with PBS to remove cell debris and unattached cells. DMEM complete medium containing 2 mg / mL of peptide was added to each well of the 12-well plate. A control group containing DMEM complete medium without peptide was set up. After sample addition, three locations in the center of the scratch in each well were photographed. Image J software was used for automated image analysis to calculate the average cell confluence area. Three photos were taken at the same location 12 hours later and Image J software was used for automated image analysis to calculate the average cell confluence area. The test results are shown in Figure 1 and Table 5.

[0200] Table 5: Healing rate of cell scratch test after 12 hours Note: *P<0.05; **P<0.01 vs Control

[0201] The results showed that the healing rates of the culture media with the addition of peptide 1, peptide 3, peptide 4, peptide 5, and peptide 6 were significantly higher than those of the blank culture media without peptide, while that of peptide 2 was slightly higher than that of the blank culture media without peptide, indicating that the peptides provided by the present invention have strong cell repair ability.

[0202] Example 5: Inhibition of anti-inflammatory activity

[0203] In order to observe the anti-inflammatory effect of the peptide provided by the present invention, an anti-inflammatory activity inhibition test was performed using HaCat cells (Mingzhou Bio, MZ-0069). The solution used in this example was prepared as follows:

[0204] Complete DMEM medium: 89% high-glucose DMEM medium (Gibco), 10% fetal bovine serum (Gibco), 1% 100× penicillin-streptomycin double antibody (Sigma-Aldrich);

[0205] Peptide solution for the experimental group: The peptide was dissolved in complete DMEM medium at a concentration of 2 mg / mL and stored at 4°C;

[0206] Inflammation inducer: fresh Escherichia coli DH5α, washed twice with PBS, resuspended in PBS, adjusted the bacterial solution to OD = 1.0, boiled at 95℃ for 10 minutes, and can be stored at -80℃.

[0207] Positive control: Palmitoyl tetrapeptide-7 (Guangzhou Baiyu Biological) was prepared into 2 mg / mL in complete DMEM medium and stored at 4°C.

[0208] The experimental process of this example is as follows: HaCat cells were seeded in a twelve-well plate, with 1×10 5 The culture medium was 0.5 mL of complete DMEM. After the cells adhered, the culture medium was discarded and fresh culture medium containing peptides 1 to 7 was added, along with 50 μL of an inflammatory inducer (in triplicate) for each peptide. A positive control consisted of culture medium supplemented with the inflammatory inducer alone, and a blank control was supplemented with 50 μL of PBS. The cells were incubated for 24 hours. The supernatant was collected and IL-8 levels were measured using a human IL-8 detection kit (Dakoway Biotechnology Co., Ltd.).

[0209] The calculation process is as follows:

[0210] Inhibition rate = 1-(C 实验组 -C 空白组 ) / (C 阳性组 -C 空白组 )

[0211] The test results are shown in Table 6:

[0212] Table 6: Supernatant IL-8 levels and inhibition rates

[0213] From the results in the above table, it can be seen that peptides 1 to 6 all have a certain inhibitory effect on the expression of IL-8 activated by Escherichia coli inflammatory inducers, among which peptide 6 has the most significant effect.

[0214] Example 6: Hydrating and moisturizing effects

[0215] The hydrating and moisturizing effects of the peptide provided by the present invention were observed using a zebrafish experiment. The solution used in this example was prepared as follows:

[0216] Peptide solution in the experimental group: The peptides were prepared into 1% with standard dilution water and used immediately after preparation.

[0217] Positive control: Sodium hyaluronate fruit-flavored drink (Jiangsu Yijiayuan Health Technology Co., Ltd.), stored in a cool, dry place. Directly aspirate the original solution and use it immediately.

[0218] The experimental process of this embodiment is as follows: 2dpf melanin allele mutant Albino strain zebrafish were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). The samples were given water-soluble (all at a concentration of 0.05%), and the positive control was a sodium hyaluronate fruit-flavored drink at a concentration of 15.6μL / mL. At the same time, a normal control group and a model control group were set up, and the capacity of each well was 3mL. Except for the normal control group, the other experimental groups were given sodium chloride in water to establish a zebrafish water deprivation model. After treatment at 28℃ for 22h, 10 zebrafish were randomly selected from each experimental group and placed under a dissecting microscope for photography. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software, and the zebrafish tail area (S) was analyzed. The statistical analysis results of this indicator were used to evaluate the hydrating and moisturizing efficacy of the samples.

[0219] The results are expressed as mean ± SE. The formula for calculating the hydrating and moisturizing effect is as follows:

[0220] Statistical analysis was performed using SPSS 26.0 software, with p < 0.05 indicating statistically significant differences. The results are shown in Figures 2 and 3 and Table 7.

[0221] Table 7: Zebrafish tail water loss test to detect the hydrating and moisturizing effect of peptides Note 1. Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001 Note 2. The higher the hydration and moisturizing rate, the stronger the effect

[0222] It can be seen from the results in the above table that peptide 1, peptide 2, peptide 3, peptide 5, and peptide 6 can all protect the zebrafish tail area reduction caused by dehydration, and have the effect of hydrating and moisturizing.

[0223] Example 7: Identification of Deinococcus microti

[0224] This embodiment discloses a method for identifying Deinococcus microti, specifically, a method for analyzing specific primers and specific sequences, including:

[0225] 1. Genomic DNA extraction

[0226] The specimens in this example were isolated from Deinococcus wulumuqiensis R12 isolated by Hangzhou Weizhi Biotechnology Co., Ltd. T , China General Microbial Culture Collection Center), D. radiodurans DSM 20539 T , China Marine Microbial Culture Collection Center), and DNA template extraction was performed using a kit provided by Guangzhou Meiji Biotechnology Co., Ltd. (product number: D3146).

[0227] 2. 16S rRNA gene sequence analysis

[0228] PCR amplification of the isolated Deinococcus was performed using the primers shown in Table 8. SEQ ID NOs: 13-24 correspond to the upstream and downstream primers of the nucleotide sequences encoding peptide 1 to peptide 6, respectively. The reaction system is shown in Table 9.

[0229] Table 8: Primer design

[0230] Table 9: Reaction system used

[0231] This experiment used NEB's High-Fidelity PCR Master Mix (Cat. #M0531) is used for PCR amplification. This master mix combines a high-fidelity polymerase with an optimized reaction buffer to ensure high amplification efficiency and accuracy in PCR reactions.

[0232] When preparing the PCR reaction, first calculate the required amount of DNA template according to the following formula: Addition volume = Total DNA amount / Concentration. The total amount of DNA should be kept within the range of 50 ng to 250 ng to ensure the specificity and efficiency of the PCR reaction. Accurately calculating the amount of DNA template added ensures uniform distribution of the DNA in the reaction system, thereby improving the reproducibility and accuracy of amplification. The amplification protocol is shown in Table 10.

[0233] Table 10: Amplification Procedure

[0234] 3. PCR product processing and sequencing

[0235] Gel electrophoresis detection:

[0236] After the PCR reaction is complete, the PCR products are first checked for purity by gel electrophoresis. Gel electrophoresis is a standard molecular biology technique used to separate and identify nucleic acid fragments. This step ensures that the PCR products are free of nonspecific amplification or contamination. As shown in Figure 4, the PCR products ranged from 249 to 382 bp in length, with the VB142 product being less than 500 bp. However, no PCR products were observed for Deinococcus urumqiensis and Deinococcus radiodurans.

[0237] Furthermore, the inventors purchased Deinococcus radiodurans DRR1, ATCC 27603, and 17438 from CCTCC and repeated the above steps, finding no PCR product from Deinococcus radiodurans. The detection procedure is as follows:

[0238] (1) Purity and concentration determination:

[0239] Use the Nanodrop spectrophotometer to accurately measure the concentration of PCR products. Nanodrop technology provides detailed information on sample concentration and purity, ensuring that the quality of the samples submitted meets sequencing requirements.

[0240] (2) Sample testing:

[0241] The PCR products that have passed the purity and concentration tests will be sent to Sangon Biotech (Shanghai) Co., Ltd. (abbreviated as Sangon Biotech) for first-generation sequencing analysis.

[0242] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0243] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and variations without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A polypeptide, characterized in that The polypeptide sequence has at least one of the amino acids shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

2. An isolated polynucleotide, characterized in that The polynucleotide encodes the polypeptide of claim 1; Optionally, the polynucleotide has at least one of the nucleotide sequences shown in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

3. An expression vector, characterized in that Carrying the polynucleotide according to claim 2.

4. A recombinant cell, characterized in that Carrying the polynucleotide of claim 2, the expression vector of claim 3, or being capable of expressing the polypeptide of claim 1.

5. The recombinant cell according to claim 4, characterized in that The recombinant cell is obtained by introducing the expression vector according to claim 3 into a host cell.

6. A composition, characterized in that Comprising at least one of the polypeptide according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3, and the recombinant cell according to claim 4 or 5.

7. A medicine for external use, characterized in that: The drug comprises at least one of the polypeptide according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3, the recombinant cell according to claim 4 or 5, and the composition according to claim 6.

8. A cosmetic for external use, characterized in that The cosmetic comprises at least one of the polypeptide according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3, the recombinant cell according to claim 4 or 5, and the composition according to claim 6.

9. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3, the recombinant cell according to claim 4 or 5, or the composition according to claim 6 in the preparation of cosmetics; Optionally, the cosmetic has at least one of the effects of anti-inflammation, scar reduction, and moisturizing.

10. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 3, the recombinant cell according to claim 4 or 5, or the composition according to claim 6 in the preparation of a medicament; Optionally, the drug has at least one of the effects of anti-inflammation, accelerating wound healing of the skin surface, and reducing scar formation.

11. Use of the polypeptide according to claim 1 or the polynucleotide according to claim 2 in preparing a detection kit for identifying Deinococcus microti.

12. A kit for detecting Deinococcus microti, characterized in that: The kit comprises the polypeptide according to claim 1 and / or the polynucleotide according to claim 2.

13. Use of the polypeptide according to claim 1 and / or the polynucleotide according to claim 2 in detecting Deinococcus microti.

14. A method for identifying microdeinococci, characterized in that: include: (1) designing a primer pair that is reverse complementary to the sequence of the polynucleotide according to claim 2; (2) using the primers to amplify the sample to be tested; or, (3) contacting the polypeptide of claim 1 with a sample to be tested; Wherein, the sample to be tested is a sample containing Deinococcus microti.

15. The method according to claim 14, characterized in that The primer pair has at least one of the following nucleotide sequences: (a) the nucleotide sequences described in SEQ ID NO: 13 and SEQ ID NO: 14; (b) the nucleotide sequences described in SEQ ID NO: 15 and SEQ ID NO: 16; (c) the nucleotide sequences described in SEQ ID NO: 17 and SEQ ID NO: 18; (d) the nucleotide sequences described in SEQ ID NO: 19 and SEQ ID NO: 20; (e) the nucleotide sequences described in SEQ ID NO: 21 and SEQ ID NO: 22; (f) The nucleotide sequences described in SEQ ID NO: 23 and SEQ ID NO: 24.