Recombinant collagen and use thereof

Abstract

The present invention relates to the technical field of collagen, and in particular to a recombinant collagen and a use thereof. The recombinant collagen comprises AXBYCZ, wherein X and Z are each independently selected from 0 or 1, and Y is an integer greater than or equal to 0; when Y is 0, X and Z are not 0 at the same time; domain A is an N-terminal telopeptide of the collagen or a peptide segment thereof, domain B is Gly-Xaa-Yaa, and domain C is a C-terminal telopeptide of the collagen or a peptide segment thereof. The recombinant collagen can not only maintain high cell viability, cell adhesion rate, and cell migration rate, but also has an anti-inflammatory effect and a melanogenesis inhibitory effect, and can be widely applied to the fields of biomedicine, medical instruments and cosmetics, and the technical field of bioengineering.

Description

A recombinant collagen and its application Technical Field

[0001] This invention relates to the field of collagen technology, specifically to a recombinant collagen and its applications. Background Technology

[0002] Collagen (or simply collagen) is a major protein that makes up the structure of human tissues, accounting for about 30-40% of the total protein in the human body. It is a major component of human tissues and organs and is also known as the "framework of life." As a medical material, collagen has been widely used in the medical and health field for the repair of human skin, bones, cartilage, cardiovascular system, oral cavity and luminal tissues, as well as in cosmetic surgery.

[0003] Currently, collagen is mainly obtained from animal skin, bones, and other tissues through acid, alkaline, and enzymatic hydrolysis. Although there are various extraction methods for collagen, animal-derived collagen is all type I and has several drawbacks, including immunogenicity, poor uniformity, and the risk of infectious diseases. More importantly, animal-derived collagen is usually obtained through acid, alkali, and enzymatic hydrolysis, and its biological activity is far inferior to that of natural human collagen.

[0004] The amino acid sequence of collagen is divided into five parts, including an N-terminal propeptide, an N-terminal telopeptide, a Gly-Xaa-Yaa repeat domain, a C-terminal telopeptide, and a C-terminal propeptide. Current research on recombinant collagen primarily focuses on the Gly-Xaa-Yaa repeat domain. For example, patent document (CN117229386A) discloses a small-molecule collagen C3T1, which exhibits good cell adhesion activity, cell migration-promoting activity, and transdermal penetration, while also showing low cytotoxicity and possessing anti-wrinkle, oil-controlling, and repair functions. However, existing technologies have limited research on the functions of the N-terminal and / or C-terminal sequences linked to the Gly-Xaa-Yaa repeat domain, particularly the C-terminal telopeptide and / or N-terminal telopeptide. How to combine the intermediate repeat region with the telopeptide functional region to construct recombinant collagen with low immunogenicity and high activity, and to develop novel recombinant collagen sequences, is a pressing technical problem in this field. Summary of the Invention

[0005] This invention provides a recombinant collagen, wherein the recombinant collagen comprises A X B Y C Z The recombinant collagen described not only possesses high cell viability, cell adhesion rate, and cell migration rate, but also exhibits anti-inflammatory effects and melanin-inhibiting effects, making it widely applicable in the fields of biomedicine, medical devices, cosmetics, and bioengineering. Specifically:

[0006] In a first aspect, the present invention provides a recombinant collagen, said recombinant collagen comprising A X B Y C Z ;

[0007] Among them, domain A is an N-terminal peptide of collagen or a peptide segment thereof, domain B is Gly-Xaa-Yaa, and domain C is a C-terminal peptide of collagen or a peptide segment thereof.

[0008] Where X and Z are independently selected from 0 or 1, and Y is an integer ≥ 0;

[0009] When X and Z are both 0, Y is not 0;

[0010] Preferably, when Y is 0, X and Z are not both 0.

[0011] More preferably, recombinant collagen includes A X B Y C Z In this case, Z = 1, and X and Y are both integers ≥ 0.

[0012] In one specific embodiment of the present invention, X, Y, and Z are selected from any one of the following groups:

[0013] i) X is 0, Y is an integer ≥ 1, and Z is 1;

[0014] ii) X is 1, Y is an integer ≥ 1, and Z is 0;

[0015] iii) X is 1, Y is an integer ≥ 1, and Z is 1;

[0016] iv) X is 1, Y is 0, Z is 1;

[0017] v) X is 0, Y is an integer ≥ 1, and Z is 0;

[0018] vi) X is 1, Y is 0, Z is 0.

[0019] In a preferred embodiment of the present invention, X, Y, and Z are selected from any group of i), iii), and iv).

[0020] The C domain is selected from one or more of the C-terminal telopeptides or peptide segments of human type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, or type X collagen.

[0021] In one specific embodiment of the present invention, the C domain is one or more of the C-terminal peptides or peptide segments of human type I collagen, type II collagen, or type III collagen.

[0022] Preferably, the C domain is the C-terminal peptide of human type III collagen or a peptide segment thereof.

[0023] Preferably, the human type III collagen C-terminal telopeptide or its peptide fragment comprises at least 1 to 25 consecutive amino acids, at least 2 to 25 consecutive amino acids, such as at least 5 to 25 consecutive amino acids, at least 5 to at least 21 consecutive amino acids, at least 5 to at least 17 consecutive amino acids, at least 5 to at least 13 consecutive amino acids, at least 5 to at least 9 consecutive amino acids, at least 9 to 25 consecutive amino acids, at least 9 to at least 21 consecutive amino acids, at least 9 to at least 17 consecutive amino acids, or at least 9 to at least 13 consecutive amino acids. At least 13 to at least 21 consecutive amino acids, at least 13 to at least 25 consecutive amino acids, at least 13 to at least 17 consecutive amino acids, at least 17 to at least 21 consecutive amino acids, at least 17 to at least 25 consecutive amino acids, at least 21 to at least 25 consecutive amino acids; human type III collagen C-terminal telopeptide or its peptide segments, for example, containing human type III collagen C-terminal telopeptide 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive amino acids.

[0024] Preferably, the human type III collagen C-terminal telopeptide or its peptide segment comprises at least 1 to 25 consecutive amino acids, at least 2 to 25 consecutive amino acids from the N-terminus, for example, at least 5 to 25 consecutive amino acids from the N-terminus, at least 5 to at least 21 consecutive amino acids from the N-terminus, at least 5 to at least 17 consecutive amino acids from the N-terminus, at least 5 to at least 13 consecutive amino acids from the N-terminus, at least 5 to at least 9 consecutive amino acids from the N-terminus, at least 9 to 25 consecutive amino acids from the N-terminus, at least 9 to at least 21 consecutive amino acids from the N-terminus, at least 9 to at least 17 consecutive amino acids from the N-terminus, at least 9 to at least 1... Three consecutive amino acids, at least 13 to at least 21 consecutive amino acids from the N-terminus, at least 13 to at least 25 consecutive amino acids from the N-terminus, at least 13 to at least 17 consecutive amino acids from the N-terminus, at least 17 to at least 21 consecutive amino acids from the N-terminus, at least 17 to at least 25 consecutive amino acids from the N-terminus, at least 21 to at least 25 consecutive amino acids from the N-terminus; human type III collagen C-terminal telopeptide or its peptide segments, for example, contain human type III collagen C-terminal telopeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive amino acids from the N-terminus.

[0025] In one specific embodiment of the present invention, the amino acid sequence of the C domain includes cysteine ​​or any one of SEQ ID NO: 56-58, 65-69.

[0026] Preferably, the amino acid sequence of the C domain includes any one of SEQ ID NO: 58, 66-69.

[0027] Domain A is selected from one or more of the N-terminal telopeptides or peptide segments of human type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, or type X collagen.

[0028] In one specific embodiment of the present invention, the A domain is one or more of the N-terminal telopeptides or peptide segments of human type I collagen, type II collagen, or type III collagen.

[0029] Preferably, the A domain is the N-terminal telopeptide of human type III collagen or a peptide segment thereof.

[0030] Preferably, the human type III collagen N-terminal telopeptide or its peptide segment comprises at least 9 to 19 consecutive amino acids, such as at least 10 to 19 consecutive amino acids, at least 11 to 19 consecutive amino acids, at least 12 to 19 consecutive amino acids, at least 13 to 19 consecutive amino acids, at least 14 to 19 consecutive amino acids, at least 15 to 19 consecutive amino acids, at least 16 to 19 consecutive amino acids, at least 17 to 19 consecutive amino acids, at least 18 to 19 consecutive amino acids, at least 13 to at least 18 consecutive amino acids, at least 13 to at least 17 consecutive amino acids, at least 14 to at least 17 consecutive amino acids, or at least 13 to at least 15 consecutive amino acids. The human type III collagen N-terminal telopeptide or its peptide segment may, for example, comprise 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive amino acids.

[0031] Preferably, the human type III collagen N-terminal telopeptide or its peptide segment comprises at least 9 to 19 consecutive amino acids from the C-terminus of the human type III collagen N-terminal telopeptide, for example, at least 10 to 19 consecutive amino acids from the C-terminus, at least 11 to 19 consecutive amino acids from the C-terminus, at least 12 to 19 consecutive amino acids from the C-terminus, at least 13 to 19 consecutive amino acids from the C-terminus, at least 14 to 19 consecutive amino acids from the C-terminus, at least 15 to 19 consecutive amino acids from the C-terminus, at least 16 to 19 consecutive amino acids from the C-terminus, and so on. At least 17 to 19 consecutive amino acids, at least 18 to 19 consecutive amino acids from the C-terminus, at least 13 to at least 18 consecutive amino acids from the C-terminus, at least 13 to at least 17 consecutive amino acids from the C-terminus, at least 14 to at least 17 consecutive amino acids from the C-terminus, at least 13 to at least 15 consecutive amino acids from the C-terminus, or human type III collagen N-terminal telopeptide or its peptide segments, for example, containing human type III collagen N-terminal telopeptide with 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 consecutive amino acids from the C-terminus.

[0032] In one specific embodiment of the present invention, the amino acid sequence of the A domain includes any one of SEQ ID NO: 53-55 and 70-77.

[0033] Preferably, the amino acid sequence of the A domain includes any one of SEQ ID NO: 55, 72-77.

[0034] Preferably, the B structural domain includes n identical or different Gly-Xaa-Yaa sequences, where n is an integer ≥ 3.

[0035] Preferably, Xaa and Yaa in the B domain can be the same or different.

[0036] Preferably, Xaa and Yaa can be any amino acid (see non-patent literature: consensus on characterization and quality evaluation of medical collagen products. China Pharmaceutical Affairs, 2019, 33(11):1223-1234.), for example, each Xaa or Yaa is independently selected from any one of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val or Hyp.

[0037] Preferably, each Xaa is independently selected from any one of Glu, Lys, Ala, Pro, Leu, or Asp.

[0038] Preferably, each Yaa is independently selected from any one of Pro, Asn, Lys, Arg, Ala, Val, Glu, or Asp.

[0039] Preferably, the B domain is selected from at least 15, 18, 21, 24, 27 or 30 consecutive amino acid sequences in the triple helix domain of human collagen.

[0040] Preferably, the amino acid sequence of the B domain includes Gly-Xaa-Yaa of human collagen;

[0041] Preferably, the human-derived collagen is one or more of type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, or type X collagen; further, the human-derived collagen is preferably one or more of type I, type II, or type III collagen.

[0042] Preferably, Y is an integer from 0 to 100, more preferably an integer from 3 to 50, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100.

[0043] In one specific embodiment of the present invention, each Xaa is independently selected from Glu, Lys, Ala or Pro, and each Yaa is independently selected from Pro, Asn, Lys, Arg or Ala.

[0044] In one specific embodiment of the present invention, each Xaa is independently selected from Glu, Ala, Leu, Pro or Asp, and each Yaa is independently selected from Arg, Ala, Pro or Lys.

[0045] In one specific embodiment of the present invention, each Xaa is independently selected from Pro, Asp, Glu, Ala or Lys, and each Yaa is independently selected from Lys, Arg, Val, Glu, Pro or Asp.

[0046] Preferably, the N-terminus of the B-domain is connected to the C-terminus of the A-domain, and the C-terminus of the B-domain is connected to the N-terminus of the C-domain.

[0047] In one specific embodiment of the present invention, the amino acid sequence of the B domain includes any one or more of SEQ ID NO: 78-80, or includes having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of the amino acid sequences shown in SEQ ID NO: 78-80.

[0048] The amino acid sequence of the recombinant collagen includes any one or more of the amino acid sequences in SEQ ID NO: 1-26, or includes amino acid sequences that have 80% or more, 85% or more, 90% or more, 95% or more or more homology with any one or more of the amino acid sequences in SEQ ID NO: 1-26.

[0049] In one specific embodiment of the present invention, the amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 1-8, 11-26, or includes having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 1-8, 11-26.

[0050] Preferably, the amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 3, 5-18, 20, 22-26, or includes amino acid sequences having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more homology with any one or more of SEQ ID NO: 3, 5-18, 20, 22-26.

[0051] In one specific embodiment of the present invention, the amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 3, 5-18, 20, 22-23, 25-26, or includes amino acid sequences having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more homology with any one or more of SEQ ID NO: 3, 5-18, 20, 22-23, 25-26.

[0052] In one specific embodiment of the present invention, the amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 3, 5-8, 11-18, 20, 22-26, or includes amino acid sequences having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more homology with any one or more of SEQ ID NO: 3, 5-8, 11-18, 20, 22-26.

[0053] In a second aspect, the present invention provides a recombinant collagen protein comprising a B domain, wherein the amino acid sequence of the B domain comprises any one or more of the amino acid sequences shown in SEQ ID NO: 78-80, or comprises having 80% or more, 85% or more, 90% or more, 95% or more or more, or 99% or more homology with any one or more of the amino acid sequences shown in SEQ ID NO: 78-80.

[0054] Preferably, the B domain in the recombinant collagen is repeated Y times, where Y is an integer from 0 to 100, preferably an integer from 3 to 50.

[0055] In one specific embodiment of the present invention, the amino acid sequence of the B domain is SEQ ID NO: 78.

[0056] Preferably, the recombinant collagen further includes an A domain and / or a C domain;

[0057] Preferably, the N end of the B domain is connected to the C end of the A domain.

[0058] Preferably, the C end of the B structure domain is connected to the N end of the C structure domain.

[0059] Preferably, the A domain is an N-terminal telopeptide or peptide segment of human collagen. For example, one or more of the N-terminal telopeptides or peptide segments of human type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, or type X collagen.

[0060] In one specific embodiment of the present invention, the A domain is one or more of the N-terminal telopeptides or peptide segments of human type I collagen, type II collagen, or type III collagen.

[0061] Preferably, the C domain is a C-terminal peptide or peptide segment of human collagen. For example, one or more of the C-terminal peptides or peptide segments of human type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, or type X collagen.

[0062] In one specific embodiment of the present invention, the C domain is one or more of the C-terminal peptides or peptide segments of human type I collagen, type II collagen, or type III collagen.

[0063] Preferably, the recombinant collagen includes A X B Y C Z .

[0064] In one specific embodiment of the present invention, the amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 1-8, 11-26, or includes having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 1-8, 11-26.

[0065] In one specific embodiment of the present invention, the amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 1-4, 11-26, or includes having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 1-4, 11-26.

[0066] In a third aspect, the present invention provides a polypeptide comprising an A domain and a C domain, wherein the A domain is an N-terminal peptide of human type III collagen or a peptide segment thereof, and the C domain is a C-terminal peptide of human type III collagen or a peptide segment thereof.

[0067] Preferably, the A structural domain is directly connected to the C structural domain or connected through Y B structural domains, wherein the B structural domains include n identical or different Gly-Xaa-Yaa sequences, where n is an integer ≥ 3 and Y is an integer ≥ 0.

[0068] Preferably, the amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 3, 5-18, 20, 22-26, or includes amino acid sequences having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more homology with any one or more of SEQ ID NO: 3, 5-18, 20, 22-26.

[0069] In one specific embodiment of the present invention, the amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 3, 5-18, 20, 22-23, 25-26, or includes amino acid sequences having 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more homology with any one or more of SEQ ID NO: 3, 5-18, 20, 22-23, 25-26.

[0070] In a fourth aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the recombinant collagen or the polypeptide described above.

[0071] Preferably, the nucleotide sequence of the nucleic acid includes any one or more of SEQ ID NO: 27-52, or includes nucleotide sequences having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 27-52.

[0072] In one specific embodiment of the present invention, the nucleotide sequence of the nucleic acid includes any one or more of SEQ ID NO: 27-34, 37-52, or includes nucleotide sequences having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 27-34, 37-52.

[0073] In one specific embodiment of the present invention, the nucleotide sequence of the nucleic acid includes any one or more of SEQ ID NO: 27-30, 37-52, or includes nucleotide sequences having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 27-30, 37-52.

[0074] In one specific embodiment of the present invention, the nucleotide sequence of the nucleic acid includes any one or more of SEQ ID NO: 29, 31-44, 46, 48-52, or includes nucleotide sequences having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 29, 31-44, 46, 48-52.

[0075] In one specific embodiment of the present invention, the nucleotide sequence of the nucleic acid includes any one or more of SEQ ID NO: 29, 31-44, 46, 48-49, 51-52, or includes nucleotide sequences having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 29, 31-44, 46, 48-49, 51-52.

[0076] In one specific embodiment of the present invention, the nucleotide sequence of the nucleic acid includes any one or more of SEQ ID NO: 29, 31-34, 37-44, 46, 48-52, or includes nucleotide sequences having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 29, 31-34, 37-44, 46, 48-52.

[0077] In one specific embodiment of the present invention, the nucleotide sequence of the nucleic acid includes any one or more of SEQ ID NO: 29, 37-42, 46, 48-49, 51-52, or includes nucleotide sequences having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% homology with any one or more of SEQ ID NO: 29, 37-42, 46, 48-49, 51-52.

[0078] In a fifth aspect, the present invention provides an expression vector comprising the above-described nucleic acid, and / or expressing the above-described recombinant collagen or the above-described polypeptide;

[0079] Preferably, the backbone of the expression vector includes a prokaryotic vector backbone or a eukaryotic vector backbone.

[0080] Preferably, the expression vector includes one or more of pET29a, pPIC9k, pPICZαA, pHIL-S1, or pYAM75P.

[0081] In a sixth aspect, the present invention provides a method for preparing the above-mentioned recombinant collagen or the above-mentioned polypeptide, wherein the preparation method includes chemical synthesis or biosynthesis;

[0082] Preferably, the biosynthesis includes prokaryotic expression or eukaryotic expression.

[0083] In one specific embodiment of the present invention, the preparation method includes:

[0084] 1) Obtain the above-mentioned nucleic acids;

[0085] 2) The nucleic acids obtained in 1) are ligated to a backbone vector to obtain an expression vector;

[0086] 3) Transform the expression vector obtained in 2) into host cells to induce expression;

[0087] Preferably, the preparation method further includes a purification step.

[0088] Preferably, the nucleic acid described in step 1) is tagged, and the tag is, for example, a histidine tag, a fusion tag, or a protease cleavage site sequence. Preferably, the tag includes a 6*His tag, a GST tag, an MBP tag, or a SUMO tag.

[0089] Preferably, step 2) includes codon optimization of the obtained nucleic acid, splicing and recombination into a backbone vector to obtain an expression vector, wherein the enzyme cleavage sites on the backbone vector preferably include Xba I and Hind III.

[0090] Preferably, the preparation method further includes obtaining collagen through enzymatic digestion.

[0091] Preferably, the host cell includes a eukaryotic cell or a prokaryotic cell.

[0092] Preferably, the eukaryotic cells include one or more of yeast (e.g., Pichia pastoris or Saccharomyces cerevisiae), animal cells, or plant cells.

[0093] Preferably, the prokaryotic cells include Escherichia coli or Bacillus (e.g., Bacillus subtilis or Bacillus licheniformis).

[0094] In one specific embodiment of the present invention, the host cell is Escherichia coli.

[0095] In a seventh aspect, the present invention provides a host cell comprising the expression vector described above.

[0096] Preferably, the host cell includes a eukaryotic cell or a prokaryotic cell.

[0097] Preferably, the eukaryotic cells include one or more of yeast (e.g., Pichia pastoris or Saccharomyces cerevisiae), animal cells, or plant cells.

[0098] Preferably, the prokaryotic cells include Escherichia coli or Bacillus (e.g., Bacillus subtilis or Bacillus licheniformis).

[0099] In one specific embodiment of the present invention, the host cell is Escherichia coli.

[0100] In an eighth aspect, the present invention provides an application of the above-described recombinant collagen, the above-described polypeptide, the above-described nucleic acid, the above-described expression vector, or the above-described host cell, wherein the application includes one or more applications in the fields of biomedicine, medical devices, cosmetics, or bioengineering.

[0101] A ninth aspect of the present invention provides an application of the above-described recombinant collagen, the above-described polypeptide, the above-described nucleic acid, the above-described expression vector, or the above-described host cell, the application comprising:

[0102] 1) Use in the preparation of medicaments for treating diseases. Preferably, the diseases include inflammation or diseases for which inhibition of melanin is beneficial for treatment.

[0103] 2) Application in the preparation of medical materials, preferably, the medical materials include one or more of the following: surgical sutures, medical sponges (e.g., collagen sponges), dressings (e.g., wound dressings, healing dressings or repair dressings), hemostatic materials, repair materials (e.g., skin repair, tissue repair), artificial organs (e.g., artificial skin, artificial bones, artificial cartilage, artificial blood vessels, artificial tendons), filler materials (e.g., skin filler materials or tissue filler materials), biological scaffolds, hydrogels, and drug carriers;

[0104] 3) Application as an active ingredient in cosmetics, food additives, health products, or animal feed, wherein the active ingredient in cosmetics preferably includes whitening or anti-inflammatory ingredients.

[0105] Preferably, the inflammation includes degenerative inflammation, exudative inflammation (e.g., one or more of serous inflammation, fibrinous inflammation, purulent inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, or specific inflammation (e.g., one or more of tuberculosis, syphilis, leprosy, or lymphogranuloma).

[0106] Preferably, the treatment of inflammation includes inhibiting inflammatory factors. Preferably, the inflammatory factors include TNF-α.

[0107] Preferably, the treatment of inflammation includes inhibiting inflammatory factors through the aforementioned recombinant collagen or the aforementioned peptide.

[0108] Preferably, the diseases for which inhibiting melanin is beneficial for treatment include pigmentation, melasma, and melanoma (e.g., acral melanoma, mucosal melanoma, chronic sun damage type (CSD), and non-chronic sun damage type).

[0109] Preferably, the diseases for which treatment is beneficial by inhibiting melanin production include those for which melanin production is inhibited by the aforementioned recombinant collagen or the aforementioned polypeptide.

[0110] In one specific embodiment of the present invention, the application includes the preparation of repair materials (e.g., skin repair, tissue repair), filler materials (e.g., skin filler, tissue filler), hemostatic materials, or dressings (e.g., wound dressings, healing dressings, or repair dressings).

[0111] In one specific embodiment of the invention, the application includes its use in the preparation of medicaments for treating diseases. Preferably, the diseases include inflammation or diseases for which inhibiting melanin production is beneficial for treatment.

[0112] In one specific embodiment of the present invention, the application includes scenarios such as whitening, skin care, repair, and anti-inflammation, for example, in the medical aesthetics or skin care product field that specifically improves or solves skin problems such as dull skin, skin aging, sagging, inflammation, wrinkles, etc.

[0113] In one specific embodiment of the present invention, the applications include the preparation of repair materials, filling materials (e.g., skin fillers, tissue fillers), hemostatic materials or dressings (e.g., wound dressings, healing dressings, etc.), applications in the preparation of drugs for treating diseases (preferably, the diseases include inflammation or diseases that are beneficial to treatment by inhibiting melanin), and applications in whitening, skin care, repair, anti-inflammatory and other scenarios.

[0114] In a tenth aspect, the present invention provides a medicament comprising the above-described recombinant collagen, the above-described polypeptide, the above-described nucleic acid, the above-described expression vector, or the above-described host cell.

[0115] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0116] Preferably, the pharmaceutically acceptable excipients include one or more of the following: excipients, diluents, lubricants, wetting agents, emulsifiers, preservatives, antioxidants, buffers, antibacterial agents, suspending agents, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, suspending agents, solubilizers, thickeners, or stabilizers.

[0117] Preferably, the formulation of the drug can be any one or a combination of two or more of the following: syrup, elixir, suspension, powder, granule, tablet, capsule, pill, lozenge, rectal suppository or rectal enema, injection, aqueous solution, cream, ointment, lotion, gel or emulsion (e.g., water-in-oil or oil-in-water emulsion).

[0118] Preferably, the formulation of the drug is a unit-dose formulation. The amount of the active pharmaceutical ingredient in the unit-dose formulation can be varied or adjusted from 0.001 mg to 1000 mg (preferably from 0.1 mg to 500 mg, more preferably from 30 mg to 100 mg, for example 0.001, 0.01, 0.1, 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 mg), depending on the specific application and efficacy of the active pharmaceutical ingredient.

[0119] Preferably, the drug is beneficial for treating inflammation or inhibiting melanin in diseases.

[0120] Depending on the specific implementation requirements, the drug may also contain other suitable therapeutic agents, such as anti-inflammatory agents or agents that inhibit melanin production.

[0121] Preferably, the drug is suitable for gastrointestinal or non-gastrointestinal administration.

[0122] Preferably, the administration route of the drug includes gastrointestinal administration (e.g., oral administration, enteral administration, or catheter administration) or non-gastrointestinal administration (e.g., topical, intravenous, intramuscular, intradermal, and subcutaneous administration).

[0123] In an eleventh aspect, the present invention provides a cosmetic product comprising the above-described recombinant collagen, the above-described polypeptide, the above-described nucleic acid, the above-described expression vector, or the above-described host cell.

[0124] Preferably, the cosmetic product further includes excipients acceptable in the cosmetics field.

[0125] Preferably, the cosmetic excipients acceptable in the field include one or more of the following: solvents, solubilizers, preservatives, antioxidants, pH adjusters, penetration enhancers, oils, humectants, thickeners, chelating agents, skin feel modifiers, surfactants, emulsifiers, amino acids, propellants / projectiles, fragrances, pigments, and other functional additives.

[0126] Preferably, the dosage form of the cosmetic includes one or more of the following: suspension, emulsion, paste, gel, cream, lotion, powder, soap, facial cleanser, liquid foundation (e.g., powder foundation, emulsion foundation, or wax foundation), mask, massage cream, or spray.

[0127] In a twelfth aspect of the present invention, a method for treating a disease is provided, the method comprising administering to a subject in need an effective amount of the recombinant collagen, the polypeptide, the nucleic acid, the expression vector, the host cell, or the drug described above.

[0128] Preferably, the administration method includes gastrointestinal administration (e.g., oral administration, enteral administration, or catheter administration) or non-gastrointestinal administration (e.g., topical, intravenous, intramuscular, intradermal, and subcutaneous administration).

[0129] Preferably, the disease includes the disease described in the ninth aspect above.

[0130] In a thirteenth aspect, the present invention provides a whitening or skincare method, the whitening or skincare method comprising using the above-described recombinant collagen, the above-described polypeptide, the above-described nucleic acid, the above-described expression vector, the above-described host cell, the above-described drug, or the above-described cosmetic.

[0131] Preferably, it achieves whitening and / or skincare effects by inhibiting melanin production.

[0132] Preferably, the whitening and / or skin care effects are achieved by inhibiting melanin production through the aforementioned collagen, polypeptide, nucleic acid, expression vector, host cell, or drug.

[0133] The "subject" described in this invention can be a human or a non-human mammal, or a cell, tissue, or organ of a human or non-human mammal. The non-human mammal can be a wild animal, a zoo animal, an economically important animal, a pet, a laboratory animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, minks, jackals, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), or monkeys, etc.

[0134] The term "effective amount" as used in this invention refers to the amount or dose of the product of this invention that provides the desired treatment after being administered to an individual or organ in a single dose or multiple doses.

[0135] The term "pharmaceutically acceptable" or "cosmetically acceptable" as used in this invention refers to the biological activity and characteristics of a pharmaceutically active substance in a product that neither significantly irritates the organism nor inhibits its activity.

[0136] The term "treatment" as used in this invention refers to delaying, terminating, blocking, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0137] The term "prevention" as used in this invention refers to all behaviors that suppress or delay specific symptoms of stress by applying the products described in this invention.

[0138] The term "homology" as used in this invention refers to the ability of those skilled in the art, when using amino acid or nucleotide sequences, to adjust the sequence according to actual working needs, while ensuring structural or functional similarity to known sequences, so that the sequence used has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, and 36% similarity to sequences obtained by existing technologies. 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the sameness.

[0139] The term "and / or" as used in this invention encompasses all combinations of items connected by the term, and should be considered as if each combination had been individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". As another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0140] The use of "comprising" or "including" in this invention is an open-ended description, encompassing the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect them.

[0141] The beneficial effects of this invention are:

[0142] This invention selects the N-terminal or C-terminal region sequence of human collagen for recombinant expression, and combines it with the amino acid sequence of the middle region to obtain recombinant collagen. Optimization of the C-terminal region can enable the recombinant collagen or peptide to exhibit significant in vitro anti-inflammatory and melanin production inhibition effects, making it effective for treatment of melanin-inhibiting and anti-inflammatory diseases, as well as in medical and skin care fields. Simultaneously, through the expression of the N- and C-terminal regions, this recombinant collagen is non-cytotoxic, exhibits good cell viability, cell proliferation rate, cell adhesion rate, antioxidant capacity, and cell migration promotion ability, demonstrating excellent tissue repair effects and making it suitable for use in various medical and skin care products.

[0143] 2) This invention screens suitable amino acid sequences in the N-terminal, C-terminal, and intermediate regions to obtain recombinant collagen that simultaneously exhibits cell proliferation rate, cell adhesion rate, antioxidant and cell migration promotion capabilities, as well as significant anti-inflammatory and melanin production inhibition effects, thus possessing extremely high application value.

[0144] 3) The recombinant collagen or polypeptide produced by this invention has very good hydrophilicity and stability. Its amino acid composition is 100% identical to the corresponding part of the amino acid sequence of natural collagen. It will not produce immune rejection or allergic reaction when applied to the human body. It can be widely used in the fields of biomedicine, medical devices, cosmetics and bioengineering technology. Attached Figure Description

[0145] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0146] Figure 1: Effects of different types of collagen telopeptides on the bioactivity of recombinant collagen;

[0147] Figure 2: Effects of C-terminal peptides of different lengths of type III collagen on the bioactivity of recombinant collagen;

[0148] Figure 3: Effects of N-terminal peptides of different lengths of type III collagen on the bioactivity of recombinant collagen;

[0149] Figure 4: Exploration of the influence of amino acid sequences of different lengths and types in the middle region on the bioactivity of recombinant collagen;

[0150] Figure 5: Exploring the reasons for the influence of type III collagen telopeptides on the bioactivity of recombinant collagen. Detailed Implementation

[0151] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0152] Example 1: Gene Design and Synthesis

[0153] 1) Gene design:

[0154] Based on the sequence characteristics of human type I collagen and human type III collagen, a recombinant collagen sequence COL-1 was designed. Its amino acid sequence is shown in SEQ ID NO: 1. Its N-terminus contains the N-terminal peptide sequence of human type I collagen, and its C-terminus contains the C-terminal peptide sequence of human type I collagen.

[0155] 2) Codon optimization

[0156] The encoding nucleic acid sequence was reverse-engineered using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for expression in the host *E. coli*. After the above optimization, the corresponding nucleic acid fragment encoding recombinant collagen was obtained, and the nucleotide sequence of the nucleic acid fragment is shown in SEQ ID NO: 27.

[0157] 3) Gene synthesis

[0158] Based on the nucleic acid sequence shown in SEQ ID NO: 27, a nucleic acid fragment encoding a recombinant collagen fragment was synthesized by BGI Genomics or constructed using PCR.

[0159] Example 2: Expression and purification of recombinant collagen

[0160] 1) Construction of expression strains

[0161] Construction of pET29a-COL: The nucleic acid fragment synthesized in Example 1 was directly ligated into the pET29a plasmid to obtain pET29a-COL. pET29a-COL was then transformed into BL21(DE3) competent cells by heat shock, plated on Kanamycin resistance plates, and single colonies picked from the plates were the expression strains.

[0162] 2) Induced expression of the target protein

[0163] Single colonies of the expression strain were picked and inoculated into test tubes containing 2.5 mL of LB medium (with added kanamycin) and incubated overnight at 37°C. The overnight culture seed culture was then transferred to 100 mL of LB liquid medium at a 1% inoculation rate and incubated at 37°C and 200 rpm until the OD value reached approximately 2–3. IPTG was then added to a final concentration of 1.5 mM for induction culture, which was carried out for 4 hours. The bacterial cells were then collected by centrifugation.

[0164] 3) Crude purification of the target protein

[0165] A 10% (wet weight of bacteria / volume of PB) bacterial suspension was prepared using PB buffer at pH 7.0. The suspension was homogenized three times under high pressure at 800 bar. The supernatant was collected by centrifugation and was used as the crude protein expression solution. It was identified by electrophoresis: competent cells BL21(DE3) without plasmid introduction were used as the induction control. After induction, the cell wall disruption control group and the constructed expression strain group were simultaneously detected. The expression location was detected by SDS-PAGE protein electrophoresis.

[0166] 4) Purification of the target protein

[0167] Equilibrate the SP column (Qianchun Biotechnology Co., Ltd., catalog number: A20504) with 5 column volumes of equilibration buffer (10 mM NaH2PO3, pH 3.0). Then load the crude protein expression solution described above onto the column to ensure the target protein is fully bound. Next, equilibrate the SP column with 5 column volumes of equilibration buffer (10 mM NaH2PO3, 500 mM NaCl, pH 3.0). Elute any remaining proteins with 5 column volumes of elution buffer (10 mM NaH2PO3, 150 mM NaCl, pH 3.0). Finally, elute the target protein with 2 column volumes of elution buffer (10 mM NaH2PO3, 500 mM NaCl, pH 3.0).

[0168] The resulting product was dialyzed overnight and lyophilized into a dry powder for later use.

[0169] Other different recombinant collagen sequences were designed and prepared in accordance with Example 1. The amino acid sequences and corresponding nucleotide sequences are shown in Table 1.

[0170] Table 1: Collagen Sequences (COL Sequences)

[0171] Note: N Ⅰ The sequence represents the N-terminal telopeptide sequence of type I collagen (amino acid sequence: SEQ ID NO: 53, nucleotide sequence encoding it: SEQ ID NO: 59); C Ⅰ The C-terminal telopeptide sequence representing type I collagen (amino acid sequence: SEQ ID NO: 56, nucleotide sequence encoding it: SEQ ID NO: 62); N Ⅱ The N-terminal telopeptide sequence representing type II collagen (amino acid sequence: SEQ ID NO: 54, nucleotide sequence encoding it: SEQ ID NO: 60); C Ⅱ The C-terminal telopeptide sequence representing type II collagen (amino acid sequence: SEQ ID NO: 57, nucleotide sequence encoding it: SEQ ID NO: 63); N Ⅲ The N-terminal telopeptide sequence representing type III collagen (amino acid sequence: SEQ ID NO: 55, nucleotide sequence encoding it: SEQ ID NO: 61); C Ⅲ The C-terminal telopeptide sequence representing type III collagen (amino acid sequence: SEQ ID NO: 58, nucleotide sequence encoding it: SEQ ID NO: 64); type I collagen 10 SEQ ID NO: 79 (repeated 10 times) indicates the source of type I collagen; type II collagen 10 SEQ ID NO: 80, repeated 10 times, indicates type II collagen source; type III collagen10 SEQ ID NO: 78, indicating the source of type III collagen, repeated 10 times; C Ⅲ-4 (SEQ ID NO: 69), C Ⅲ-8 (SEQ ID NO: 68), C Ⅲ-12 (SEQ ID NO: 67), C Ⅲ-16 (SEQ ID NO: 66), C Ⅲ-20 (SEQ ID NO: 65), C Ⅲ-24 (C) represents the C-terminal telopeptide sequences of type III collagen, which are reduced by 4, 8, 12, 16, 20, and 24 amino acids respectively from the C-terminus to the N-terminus; N Ⅲ-1 (SEQ ID NO: 77), N Ⅲ-2 (SEQ ID NO: 76), N Ⅲ-3 (SEQ ID NO: 75), N Ⅲ-4 (SEQ ID NO: 74), N Ⅲ-5 (SEQ ID NO: 73), N Ⅲ-6 (SEQ ID NO: 72), N Ⅲ-8 (SEQ ID NO: 71), N Ⅲ-10 (SEQ ID NO: 70) represent the N-terminal telopeptide sequences of type III collagen that are reduced by 1, 2, 3, 4, 5, 6, 8, and 10 amino acids respectively from the N-terminus to the C-terminus.

[0172] Recombinant collagen performance testing methods:

[0173] The recombinant collagen sequences designed according to Table 1 of Example 1 were used to construct and prepare the corresponding lyophilized collagen products COL-1 to COL-26 according to the method of Example 2. Cytotoxicity, cell adhesion, cell proliferation, cell migration, in vitro anti-inflammatory, and melanin production inhibition experiments of the recombinant collagen were then performed. The specific testing methods for these experiments are as follows:

[0174] 1. Recombinant collagen cytotoxicity assay

[0175] Following the method requirements of GB / T16886.5-2017, the potential cytotoxic effects of recombinant collagen were tested using in vitro cultured mammalian L929 cells.

[0176] 1) Experimental materials

[0177] Mouse fibroblasts (L929), MEM medium, PBS, Hank balanced salt solution (HBSS), penicillin and fetal bovine serum (FBS), 0.25% trypsin (containing EDTA), isopropanol, MTT assay kit, high-density polyethylene, and DMSO.

[0178] The experimental group was selected from recombinant collagen samples from COL-1 to COL-26.

[0179] The control groups included a blank control group, a negative control group (high-density polyethylene), and a positive control group (DMSO).

[0180] 2) Experimental Procedure

[0181] Prepare 100 μl of cell suspension in a 96-well plate. Pre-incubate the plate in an incubator for 24 hours (37°C, 5% CO2). Add different concentrations of the above-mentioned experimental and control groups to the plate. Incubate the plate in an incubator for 24 hours. Then add 50 μl of MTT solution to each well and incubate for 2 hours. Discard the MTT solution, add 100 μl of isopropanol solution to each well, shake the plate, and measure the absorbance at 570 nm (reference wavelength 650 nm) using a microplate reader.

[0182] 3) Formula for calculating cell viability: Cell viability (%) = (Average OD value of experimental group / Average OD value of blank control group) × 100%

[0183] Generally speaking, cells with a viability of 70% or higher are considered to be non-cytotoxic and safe for humans; however, if cell viability drops below 70%, they may have potential cytotoxicity.

[0184] The results showed that the cell viability was 100% in the blank control group, 1.82% in the positive control group, and 98.2% in the negative control group. Cells in the blank and negative control groups (high-density polyethylene) maintained normal morphology throughout the experiment and showed no cytotoxic reaction. The positive control group (DMSO) showed severe cytotoxicity, indicating the experiment was successful. Specific experimental results are shown in Examples 3-7.

[0185] 2. Recombinant collagen cell adhesion activity experiment

[0186] 1) Experimental materials

[0187] Normal culture of NIH / 3T3 cells, MEM medium, PBS, Hank's balanced salt solution (HBSS), penicillin and streptomycin and fetal bovine serum (FBS), 0.25% trypsin (containing EDTA), DMSO, and MTT assay kit.

[0188] Take COL-1 to COL-26 and the control group (bovine serum albumin (BSA) solution), and then dilute them with PBS (pH 7.4) to 0.5 mg / mL.

[0189] 2) Experimental Procedure

[0190] Add 100 μL of COL-1 to COL-26 solution and bovine serum albumin solution (control group) to each 96-well cell culture plate, setting up 3 replicates per group. Incubate at room temperature for 60 min; then add 1×10⁻⁶ COL-1 to each well. 5 NIH / 3T3 cells in good culture condition were incubated at 37°C and 5% CO2 for 60 min. Cells were washed four times with PBS. The absorbance at 570 nm was measured using an MTT assay kit (refer to the manufacturer's instructions for specific procedures).

[0191] The higher the cell adhesion rate, the more cells the protein adheres to. The more quickly recombinant collagen can help cells adhere to the cell wall or the extracellular matrix, the better it is for building a suitable extracellular environment.

[0192] The cell adhesion rate of bovine serum albumin in the control group was 100%, indicating that the experiment was successful. For specific experimental group results, please refer to Examples 3-7.

[0193] 3. Recombinant collagen cell proliferation experiment

[0194] 1) Experimental materials

[0195] Mouse fibroblasts (L929), MEM medium, PBS, Hank balanced salt solution (HBSS), penicillin and fetal bovine serum (FBS), 0.25% trypsin (containing EDTA), isopropanol, and MTT assay kit.

[0196] The experimental group was selected from recombinant collagen samples from COL-1 to COL-26.

[0197] The control group includes a blank control group and a positive control group (complete culture medium).

[0198] 2) Experimental Procedure

[0199] The cell concentration was 4×10 4100 μl of L929 cell suspension per well was added to each 96-well plate, for a total of 4000 cells / well. The plates were incubated at 37°C and 5% CO2 for 24 hours. After incubation, the culture medium was discarded, and 1640 g starvation medium was added for another 24 hours. Following this, experimental groups, blank control group, positive control group, and COL-1 to COL-26 test samples were added to each well (6 wells per group). These were then incubated at 37°C and 5% CO2 for 48 hours. After incubation, the cell culture plates were removed, the culture medium was discarded, and 50 μl of 1 mg / ml MTT solution was added to each well. The plates were then incubated at 37°C and 5% CO2 for 2 hours.

[0200] After the culture was completed, the liquid in the culture plate was discarded, 100 μl of isopropanol was added to each well, the plate was shaken to mix, and the plate was placed in a microplate reader. The readings were taken at wavelengths of 570 nm and 650 nm. The relative cell viability of each experimental group was calculated by comparing the OD value of the blank control group. The blank control group was recorded as 100%.

[0201] 3) Results Evaluation

[0202] The OD values ​​of each group were measured, and the cell proliferation rate was calculated according to the following formula. A cell proliferation rate ≥100% was considered to have a proliferative effect.

[0203] Cell proliferation rate (%) = (average OD value of the test group / average OD value of the blank control group) × 100%.

[0204] The results showed that the cell proliferation rate of the positive control group was 130%, confirming the validity of the experiment. For specific experimental group results, please refer to Examples 3-7.

[0205] 4. Recombinant collagen promotes cell migration experiment

[0206] 1) Experimental materials

[0207] Mouse fibroblasts (L929), MEM medium, PBS, Hank balanced salt solution (HBSS), penicillin and fetal bovine serum (FBS), 0.25% trypsin (containing EDTA).

[0208] The experimental group was selected from recombinant collagen samples from COL-1 to COL-26.

[0209] 2) Experimental Procedure

[0210] ①Cell culture

[0211] First, use a marker to mark each hole on the back of the 6-hole board by dividing it into three equal parts horizontally and vertically. Mark each hole approximately (5-15) x 10 mm. 5 Seed cells. Three replicate wells per group.

[0212] ② Scratch test

[0213] Using a marker pen and a ruler, draw three horizontal lines on the bottom of the well plate. After culturing the cells for 24 hours, use a 10 μL pipette tip, aligned vertically with the ruler (or a special cell culture dish for scratching), to gently push downwards along the vertical lines to create scratches. Wash the cells three times with PBS to remove the scratched cells. Add 2 mL of serum-free medium containing COL-1 to COL-26 recombinant collagen samples to the experimental group, bringing the final concentration to 0.5 mg / mL. The blank control group only received serum-free medium.

[0214] The cells were incubated at 37°C in a 5% CO2 incubator. After 0h and 24h, images were taken under a 40x microscope with the intersection of the horizontal and vertical streaks as the core. Nine images were obtained from each well, and a total of 27 data points were collected for each combination.

[0215] ③ Data processing

[0216] The area of ​​the scratched region was measured, and the cell migration rate of each group was calculated by dividing the total area of ​​the migrating cells in the fixed scratched region by the initial area of ​​the fixed scratched region. The specific experimental group results are shown in Examples 3-7.

[0217] 5. In vitro anti-inflammatory effect study of recombinant collagen

[0218] 1) Experimental materials

[0219] Mouse macrophages (RAW264.7 cells), DMEM (high glucose) medium, PBS, Hank balanced salt solution (HBSS), penicillin and fetal bovine serum (FBS), 0.25% trypsin (containing EDTA), Mouse TNF-α ELISA Kit, bacterial lipopolysaccharide (LPS), and dexamethasone.

[0220] The experimental group was selected from recombinant collagen samples from COL-1 to COL-26.

[0221] 2) Experimental Procedure

[0222] Take well-grown RAW264.7 cells and administer them at a ratio of 1×10⁻⁶. 4 RAW264.7 cells were seeded into 96-well plates, and 100 μl of the above-mentioned recombinant collagen COL-1 to COL-26 working solution was added to each well. After the cells were seeded, they were placed in a carbon dioxide incubator and cultured for 24 h. The culture medium in the cell culture wells was then discarded.

[0223] Add medium containing LPS (final concentration 1 μg / ml) to the negative control wells. Add medium containing dexamethasone (final concentration 100 μg / ml) and LPS (final concentration 1 μg / ml) to the positive control wells. Add DMEM starvation medium to the blank control wells. Add 100 μl to each well, with two replicates per group. After drug administration, incubate the 96-well plate in a CO2 incubator for 24 h. See Table 2 for specific drug administration methods.

[0224] Table 2: Induction Drug Administration Methods

[0225] After incubation, collect 100 μl of cell culture supernatant into a 1.5 ml sterile centrifuge tube and perform the detection according to the instructions of the TNF-α ELISA kit.

[0226] 3) Calculation of TNF-α content

[0227] Plotting the concentration of the standard on the ordinate, the OD value (OD) 450 -OD 570 Using the x-axis as the horizontal axis, a standard curve regression equation was constructed. The OD values ​​of the samples were substituted into the equation to calculate the TNF-α content of the samples. Finally, the average value of each group was taken as the final TNF-α content result.

[0228] Calculation formula:

[0229] In the formula: T is the average TNF-α content of the experimental group;

[0230] C represents the average TNF-α content in the negative control.

[0231] The results showed that the inhibition rate of TNF-α content in the positive control group (59%) was above 20%, while the result in the blank control group was 30%, indicating that the experiment was successful. For specific experimental group results, please refer to Examples 3-7.

[0232] 6. Melanin Production Inhibition Test

[0233] 1) Experimental materials

[0234] Mouse melanoma cells (B16), DMEM (high glucose) medium, PBS, Hank balanced salt solution (HBSS), penicillin and fetal bovine serum (FBS), 0.25% trypsin (containing EDTA), dimethyl sulfoxide (DMSO), and thiazolyl blue (MTT) assay kit.

[0235] The experimental group was selected from recombinant collagen samples from COL-1 to COL-26.

[0236] 2) Experimental Procedure

[0237] Well-grown B16 cells were passaged into 6 mm Petri dishes and seeded at a density of 250,000 cells / well, with a volume of 4 ml per well. The dishes were then incubated in a CO2 incubator for 24 hours, after which the culture medium was discarded. The experimental group used DMEM complete medium to prepare the COL collagen sample, while the control group used DMEM complete medium. Culture conditions: 37℃, 5% CO2.

[0238] Add 4 ml of experimental group (concentration 2 mg / mL), blank group culture medium, and commonly used tyrosinase inhibitor kojic acid solution (1 mg / mL) as a control group, respectively. Incubate the 6-well plates at 37℃ for further culture. Stop culture at 48 h, collect the supernatant, centrifuge at 4000 rpm for 10 min, add 500 μl of trypsin to the cells, incubate for 2 min, collect cells with 1 ml of PBS, centrifuge at 4000 rpm for 10 min, add 500 μl of NaOH solution (1 mol / L) containing 10% DMSO to thoroughly agitate the precipitate, and incubate at 80℃ for 1 h to completely dissolve the melanin granules. Transfer 150 μl of the solution to a 96-well plate and measure the OD using a microplate reader. 390 The absorbance value at that location.

[0239] 3) Calculation of relative melanin content: Relative melanin content = OD of experimental group 390 Values / Blank Group OD 390 Value × 100%

[0240] The results showed that the melanin production inhibition rate of kojic acid solution was above 20% (49.6%), indicating that the experiment was successful. For specific experimental group results, please refer to Examples 3-7.

[0241] Example 3: Bioactivity of different types of collagen telopeptides on recombinant collagen

[0242] The COL collagen samples in Table 3 were subjected to bioactivity tests according to test methods 1 to 6. The experimental results are shown in Table 3 and Figure 1.

[0243] Table 3. Effects of different types of collagen telopeptides on the bioactivity of recombinant collagen.

[0244] As shown in Table 3 and Figure 1, the cell viability of the four recombinant collagen samples was greater than 90%, and the cell adhesion rate and cell proliferation rate were both greater than 100%, indicating that the recombinant collagen has good cell viability, cell adhesion, and cell proliferation, and has a good effect on promoting cell proliferation and adhesion. The cell migration rates of the four recombinant collagens were 38%, 32%, 82%, and 25%, respectively, indicating that all four recombinant collagens have certain cell migration-promoting properties. In particular, the cell migration rate of COL-3 (i.e., recombinant collagen using type III collagen telopeptides, SEQ ID NO: 3) was significantly higher than that of the other recombinant collagen samples.

[0245] Furthermore, COL-3 exhibited significant anti-inflammatory effects, with a TNF-α inhibition rate of 56% (TNF-α is an important inflammatory mediator, and its reduction indicates that COL-3 can effectively alleviate the inflammatory response). In contrast, COL-4 (without telopeptides), COL-1 (using type I collagen telopeptides), and COL-2 (using type II collagen telopeptides) showed anti-inflammatory rates of less than 5%, indicating weak anti-inflammatory effects. This suggests that the telopeptide structure of collagen has a certain influence on its anti-inflammatory properties, and collagen samples with type III collagen telopeptides perform exceptionally well in anti-inflammatory applications.

[0246] COL-3 also showed a significant effect in inhibiting melanin production, with an inhibition rate of 31%. In contrast, COL-4, COL-1, and COL-2 not only failed to inhibit melanin production but even promoted it. This indicates that type III collagen telopeptides play a unique role in regulating melanin production.

[0247] In summary, recombinant collagen samples using different types of collagen telopeptides all exhibited good cell activity, cell proliferation, and cell adhesion, meeting the needs of medical applications (such as medical dressings, sponges, and tissue repair). Among them, recombinant collagen using type III collagen telopeptides (COL-3) showed significant advantages in promoting cell migration, in vitro anti-inflammatory effects, and inhibiting melanin production, demonstrating marked superiority over recombinant collagen without telopeptides or using other types of collagen telopeptides (types I and II). This makes it particularly suitable for whitening and anti-inflammatory medical devices, drugs, or skincare products.

[0248] Example 4: Bioactivity of recombinant collagen with C-terminal peptides of different lengths of type III collagen

[0249] COL collagen, designed according to the amino acid sequence in Table 1 of Example 1, was constructed and prepared according to the method in Example 2 to obtain the corresponding lyophilized collagen products COL-3, COL-5 to COL-10. Bioactivity tests were performed according to the above test methods 1 to 6. The experimental results are shown in Table 4 and Figure 2.

[0250] Table 4. Effects of C-terminal peptides of different lengths of type III collagen on the bioactivity of recombinant collagen.

[0251] With the amino acid content in the middle and N-terminal regions unchanged, the effect of C-terminal amino acid truncation on collagen activity was examined. Table 4 and Figure 2 show that the cell viability of the recombinant collagen samples gradually decreased with the reduction in the number of C-terminal amino acids. This indicates that the C-terminal amino acid sequence is crucial for maintaining normal cell function and survival. When the number of C-terminal amino acids was reduced by 12 to 16, the downward trend in cell viability stabilized, and the viability still reached 70%, indicating that high cell viability was maintained. However, when the number of C-terminal amino acids was further reduced (20 or more), cell viability dropped below 65% (below 70%), suggesting that collagen may begin to have a negative impact on cells at this point.

[0252] The cell proliferation rate of recombinant collagen samples also showed the same trend as cell viability. When the number of C-terminal amino acids was reduced by less than 20, the cell proliferation rate of the samples was greater than or close to 100%, indicating that the recombinant collagen samples had a certain cell proliferation effect and the proliferation ability was relatively stable. However, when the number of C-terminal amino acids was reduced to 24, the cell proliferation rate decreased significantly to 38%.

[0253] Similarly, cell adhesion rate and cell migration rate showed the same trend as cell viability and cell proliferation rate. Therefore, when the number of C-terminal amino acids was reduced to within 0-16, collagen samples all exhibited good cell viability, cell proliferation rate, cell adhesion rate, and cell migration rate.

[0254] Furthermore, as the number of C-terminal amino acids decreased, the in vitro anti-inflammatory effect and melanin production inhibition rate of the recombinant collagen samples both showed an increasing trend. When the number of amino acids decreased by 12 to 16, the changes in the in vitro anti-inflammatory effect and melanin production inhibition rate tended to stabilize. However, if the number of C-terminal amino acids was further reduced (to 20-24), the in vitro anti-inflammatory effect and melanin production inhibition rate would drop sharply. Therefore, in order to optimize the performance of collagen samples, the number of amino acids in the C-terminal region can be controlled.

[0255] In summary, when the number of C-terminal amino acids reduced is within 0-16 (including SEQ ID NO: 58, 66-69), recombinant collagen samples exhibit good cell viability, cell proliferation, cell adhesion, cell migration, and in vitro anti-inflammatory and melanin production inhibition capabilities. Among these, when the number of C-terminal amino acids reduced is 4-16 (including SEQ ID NO: 66-69), the in vitro anti-inflammatory effect and melanin production inhibition ability are the strongest, making recombinant collagen more suitable for use in whitening and anti-inflammatory related products.

[0256] Example 5: Bioactivity of recombinant collagen with N-terminal peptides of different lengths of type III collagen

[0257] COL collagen, designed according to the amino acid sequence in Table 1 of Example 1, was constructed and prepared according to the method in Example 2 to obtain the corresponding lyophilized collagen products COL-8, COL-11 to COL-18. Bioactivity tests were performed according to test methods 1 to 6. The experimental results are shown in Table 5 and Figure 3.

[0258] Table 5. Effects of N-terminal peptides of different lengths of type III collagen on the bioactivity of recombinant collagen.

[0259] The effect of N-terminal amino acid truncation on collagen activity was investigated while keeping the amino acid content in the middle and C-terminal regions unchanged. Table 5 and Figure 3 show that truncation of the N-terminal amino acids improved cell viability, cell adhesion rate, cell proliferation rate, and cell migration rate of the recombinant collagen samples. When the number of N-terminal amino acids decreased by 0–10, the recombinant collagen samples exhibited high cell viability, proliferation activity, and adhesion properties, showing an increasing trend with a reduction of 1–5 amino acids. When 5–6 amino acids were reduced from the N-terminus, the changes in all indicators tended to stabilize. Further reduction of amino acids (8–10 amino acids) led to a decreasing trend in all indicators, but they still maintained a relatively high level.

[0260] Furthermore, both the in vitro anti-inflammatory effect and the melanin production inhibition rate decreased with the reduction of N-terminal amino acids. When the number of N-terminal amino acids decreased by 5-6, the changes in the in vitro anti-inflammatory and melanin production inhibition rates of collagen samples tended to stabilize and remained at a high level. However, when the number of N-terminal amino acids was further reduced, the in vitro anti-inflammatory and melanin production inhibition rates showed a downward trend.

[0261] In summary, reducing the number of N-terminal amino acids can significantly improve the cell viability, cell adhesion, cell proliferation, and cell migration of collagen, but it also leads to a decrease in in vitro anti-inflammatory effects and melanin production inhibition. When the number of N-terminal amino acids is reduced by 0–10 (including SEQ ID NO: 55, 70-77), the recombinant collagen samples exhibit good cell viability, cell adhesion, cell proliferation, and cell migration, making them suitable for medical applications such as dressings, hemostasis, tissue repair, and tissue filling. When the number of N-terminal amino acids is reduced by 0–6 (including SEQ ID NO: 55, 72-77), in addition to the above activities, it also shows significant in vitro anti-inflammatory effects and melanin production inhibition, making it suitable for whitening, anti-inflammatory, and other medical and cosmetic applications, thus meeting the diverse needs for recombinant collagen.

[0262] Example 6: Bioactivity of recombinant collagen with amino acid sequences of different lengths and types in the intermediate region

[0263] COL collagen, designed according to the amino acid sequence in Table 1 of Example 1, was constructed and prepared according to the method in Example 2 to obtain the corresponding lyophilized collagen products COL-4, COL-15, COL-19 to COL-23, and COL-26. Bioactivity tests were performed according to test methods 1 to 6. The experimental results are shown in Table 6 and Figure 4.

[0264] Table 6. Effects of amino acid sequences of different lengths and types in the intermediate region on the bioactivity of recombinant collagen.

[0265] To investigate the effects of the N-terminal, C-terminal, and intermediate amino acid sequences on collagen activity, this study tested the performance of recombinant collagen samples without type III N- and C-terminal sequences, without the intermediate region, and with amino acids replaced in the intermediate region, based on COL15. Table 6 and Figure 4 show that all recombinant collagen samples exhibited high cell viability (>85%), cell adhesion rate (>100%), and cell proliferation rate (>130%).

[0266] Compared with simple intermediate region collagen sequences (COL-4, COL-19, COL-21), the addition of type III collagen telopeptides (COL-15, COL-20, COL-22) significantly improved the cell migration ability of recombinant collagen samples. Moreover, the anti-inflammatory effect and the effect of inhibiting melanin production of collagen were also significantly improved, indicating that telopeptides have a certain influence on the performance of collagen.

[0267] By changing the amino acid sequences of the intermediate region to different types, using type III (COL15), type I (COL20), and type II (COL22) respectively, the anti-inflammatory effects of each group of collagen were 60%, 50%, and 50%, respectively, and the melanin production inhibition rates were 41%, 40%, and 40%, respectively. Their melanin inhibition effects are close to those of kojic acid (kojic acid can effectively inhibit tyrosinase and chelate copper ions, thereby reducing melanin production, and is a substance commonly used in the prior art to inhibit melanin), indicating that COL15, COL20, and COL22 all have good anti-inflammatory and melanin production inhibition capabilities.

[0268] Furthermore, compared to COL-15, even after shortening the amino acid sequence length in the middle region, collagen COL-23 (repeated 3 times) still maintained a high level of anti-inflammatory effect (50%) and melanin production inhibition rate (43%).

[0269] Furthermore, compared to COL-15, when the amino acid sequence in the middle region was completely removed, the anti-inflammatory effect (57%) and melanin production inhibition rate (36%) of recombinant collagen COL-26 could still be maintained at a good level.

[0270] In summary, when the amino acid sequence of the telopeptide is appropriately truncated, the recombinant collagen samples (COL15, COL20, COL22, COL-23 and COL-26) of this application all exhibit good biological activity, especially good effects in promoting cell migration, anti-inflammation and inhibiting melanin production.

[0271] Example 7: Functional study of N-terminal and C-terminal telopeptides of type III collagen

[0272] COL collagen, designed according to the amino acid sequence in Table 1 of Example 1, was constructed and prepared according to the method in Example 2 to obtain the corresponding lyophilized collagen products COL-23 to COL-25. Bioactivity tests were performed according to test methods 1 to 6. The experimental results are shown in Table 7 and Figure 5.

[0273] Table 7. Effects of telopeptides on the bioactivity of recombinant collagen.

[0274] As can be seen from Table 7 and Figure 5, the recombinant collagen prepared in this embodiment all have high cell viability (>70%), cell adhesion rate (>100%), cell proliferation rate (>100%), and cell migration rate.

[0275] Furthermore, compared to recombinant collagen (COL-23) containing both N-terminal and C-terminal telopeptides, recombinant collagen (COL-24) containing only N-terminal telopeptides showed a decrease in in vitro anti-inflammatory effect and melanin production inhibition rate, from 50% and 43% to 20% and 20%, respectively. In contrast, recombinant collagen (COL-25) containing only C-terminal telopeptides maintained a high level of in vitro anti-inflammatory effect (55%) and melanin production inhibition rate (36%). This indicates that C-terminal telopeptides have a significant impact on the anti-inflammatory and melanin production inhibition functions of recombinant collagen.

[0276] Therefore, recombinant collagen containing C-terminal peptides of type III collagen showed significant advantages in both in vitro anti-inflammatory effects and melanin inhibition. Furthermore, considering COL4 and COL23-COL26, it can be seen that collagen containing only N-terminal telopeptides also exhibits certain anti-inflammatory and melanin-inhibiting effects, indicating that N-terminal telopeptides can also endow recombinant collagen with certain anti-inflammatory and melanin-inhibiting properties.

[0277] In summary, by selecting appropriate types of N-terminal peptides, C-terminal telopeptides, truncation numbers, and suitable amino acid sequences in the intermediate region, this application can yield recombinant collagen with superior biological activity. Using appropriate N-terminal telopeptides (such as type III) and truncation numbers (such as 0-10 amino acids, preferably 0-6) is beneficial for improving the cell proliferation, adhesion, and migration effects of collagen products, making them suitable for medical or medical device products such as dressings that promote tissue growth and tissue repair. Controlling appropriate C-terminal peptides (such as type III) and truncation numbers (such as 0-16 amino acids, preferably 4-16) is beneficial for improving the anti-inflammatory and melanin-inhibiting effects of collagen products, making them suitable for whitening, anti-inflammatory, and other medical, medical device, or skincare product fields.

[0278] The recombinant collagen proteins SEQ ID NO: 3, 5-8, 11-18, 20, 22-26 of this application can achieve both of the above-mentioned effects, and at the same time have excellent properties of promoting cell proliferation, promoting cell adhesion and migration, anti-inflammation and inhibiting melanin production. They can be applied to application scenarios such as promoting repair, whitening, and anti-inflammation, and meet the needs of use.

[0279] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0280] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A recombinant collagen protein, characterized in that, The recombinant collagen includes A X B Y C Z ; Where X and Z are independently selected from 0 or 1, and Y is an integer ≥ 0; When Y is 0, X and Z are not both 0 at the same time; Domain A is an N-terminal peptide of collagen or a peptide segment thereof, preferably an N-terminal peptide of human type I, II or III collagen or a peptide segment thereof. The B domain is Gly-Xaa-Yaa; The C domain is a C-terminal peptide of collagen or a peptide segment thereof, preferably a C-terminal peptide of human type I, II or III collagen or a peptide segment thereof.

2. The recombinant collagen according to claim 1, characterized in that, X, Y, and Z are selected from any of the following groups: i) X is 0, Y is an integer ≥ 1, and Z is 1; ii) X is 1, Y is an integer ≥ 1, and Z is 0; iii) X is 1, Y is an integer ≥ 1, and Z is 1; iv) X is 1, Y is 0, Z is 1; v) X is 0, Y is an integer ≥ 1, and Z is 0; vi) X is 1, Y is 0, Z is 0.

3. The recombinant collagen according to claim 1 or 2, characterized in that, The amino acid sequence of the C domain contains at least 1 to 25 consecutive amino acids of human type III collagen C-terminal telopeptide, preferably at least 2 to 25 consecutive amino acids, and more preferably at least 5 to 25 consecutive amino acids; Preferably, the amino acid sequence of the C domain comprises at least 1 to 25 consecutive amino acids from the N-terminus of the human type III collagen C-terminal peptide, more preferably at least 2 to 25 consecutive amino acids from the N-terminus, and even more preferably at least 5 to 25 consecutive amino acids from the N-terminus.

4. The recombinant collagen according to claim 1, characterized in that, The amino acid sequence of the C domain is selected from cysteine ​​or any one of SEQ ID NO: 56-58, 65-69.

5. The recombinant collagen according to claim 1, characterized in that, The amino acid sequence of the A domain contains at least 9 to 19 consecutive amino acids of the N-terminal telopeptide of human type III collagen. Preferably, the amino acid sequence of the A domain contains at least 9 to 19 consecutive amino acids from the C-terminus of the N-terminal telopeptide of human type III collagen.

6. The recombinant collagen according to claim 5, characterized in that, The amino acid sequence of domain A includes any one of SEQ ID NO: 53-55 and 70-77.

7. The recombinant collagen according to claim 1, characterized in that, The amino acid sequence of the B domain includes Gly-Xaa-Yaa from human collagen; The human-derived collagen is preferably one or more of type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, or type X collagen. Furthermore, the human-derived collagen is preferably one or more of type I collagen, type II collagen, or type III collagen.

8. The recombinant collagen according to claim 1, characterized in that, Y is an integer from 0 to 100, preferably an integer from 3 to 50; Preferably, the amino acid sequence of the B domain includes one or more of the amino acids in SEQ ID NO: 78-80.

9. The recombinant collagen according to any one of claims 1-8, characterized in that, The amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 1-26.

10. The recombinant collagen according to claim 9, characterized in that, The amino acid sequence of the recombinant collagen includes any one or more of SEQ ID NO: 3, 5-8, 11-18, 20, 22-26.

11. A nucleic acid, characterized in that, The nucleic acid comprises a nucleotide sequence encoding the recombinant collagen of any one of claims 1-10.

12. An expression carrier, characterized in that, The expression vector comprises the nucleic acid of claim 11, and / or the expression vector expresses the recombinant collagen of any one of claims 1-10; Preferably, the backbone of the expression vector includes a prokaryotic vector backbone or a eukaryotic vector backbone.

13. A host cell, characterized in that, The host cell comprises the expression vector of claim 12; Preferably, the host cell includes a eukaryotic cell or a prokaryotic cell; More preferably, the eukaryotic cells include one or more of yeast (e.g., Pichia pastoris or Saccharomyces cerevisiae), animal cells, or plant cells; More preferably, the prokaryotic cells include Escherichia coli or Bacillus (e.g., Bacillus subtilis or Bacillus licheniformis).

14. A method for preparing recombinant collagen according to any one of claims 1-10, characterized in that, The preparation methods include chemical synthesis or biosynthesis; Preferably, the biosynthesis includes prokaryotic expression or eukaryotic expression.

15. The preparation method according to claim 14, characterized in that, include: 1) Obtain the nucleic acid as described in claim 11; 2) The nucleic acids obtained in 1) are ligated to a backbone vector to obtain an expression vector; 3) Transform the expression vector obtained in 2) into host cells and induce expression.

16. The application of any one of the recombinant collagen according to claims 1-10, the nucleic acid according to claim 11, the expression vector according to claim 12, or the host cell according to claim 13, characterized in that, The applications include: 1) Use in the preparation of medicaments for treating diseases; preferably, the diseases include inflammation or diseases for which inhibition of melanin is beneficial for treatment; 2) Application in the preparation of medical materials, preferably, the medical materials include one or more of the following: surgical sutures, medical sponges, medical dressings, hemostatic materials, artificial organs, filling materials, biological scaffolds, and drug carriers; 3) Used as an active ingredient in cosmetics, food additives, health products, or animal feed; The active ingredients in the cosmetic product preferably include whitening or anti-inflammatory ingredients.

17. A drug or cosmetic, characterized in that, The drug or cosmetic described herein includes the recombinant collagen as described in any one of claims 1-10, the nucleic acid as described in claim 11, the expression vector as described in claim 12, or the host cell as described in claim 13; Preferably, the drug is beneficial for treating inflammation or inhibiting melanin in diseases.

18. A method for treating a disease, characterized in that, The method includes administering to a subject in need an effective amount of any of the recombinant collagen of claims 1-10, the nucleic acid of claim 11, the expression vector of claim 12, the host cell of claim 13, or the drug of claim 17; Preferably, the disease includes inflammation or a disease for which inhibiting melanin production is beneficial for treatment.

19. A whitening or skincare method, characterized in that, The whitening or skincare methods described herein include the use of any of the recombinant collagen as described in claims 1-10, the nucleic acid as described in claim 11, the expression vector as described in claim 12, the host cell as described in claim 13, and the cosmetic as described in claim 17.

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