Type III collagen fusion protein as well as preparation method and application thereof

Abstract

The invention relates to a fusion protein, in particular, the fusion protein comprises the following elements fused together: (a) Annexin A5; and (b) a type III collagen. The fusion protein disclosed by the invention has the anti-inflammatory and anti-coagulation activity of annexin A5 and the repair promoting and anti-aging functions of III-type collagen, and has excellent stability, biocompatibility and application performance.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a type III collagen fusion protein, its preparation method, and its applications. Background Technology

[0002] Due to their high activity, strong specificity, and good skin compatibility, peptides have become a research hotspot and application trend in the cosmetics field in recent years, especially in the areas of whitening, moisturizing, repairing, firming, and anti-wrinkle effects. They exhibit significant efficacy potential by intervening in specific skin biological pathways, such as inhibiting tyrosinase activity, promoting collagen synthesis, and repairing the skin barrier.

[0003] Currently, technological development in this field exhibits three main characteristics: peptide structure modification, synergistic effects through complex formulations, and novel delivery systems. Regarding structural modification, peptides can be effectively enhanced in stability and bioactivity through modifications such as amidation and acetylation. For example, amidated peptides demonstrate superior antioxidant and tyrosinase inhibitory activity. In terms of synergistic effects through complex formulations, researchers are dedicated to exploring combinations of various peptides and other active ingredients to achieve a "1+1>2" effect. Existing studies have shown that specific combinations of pentapeptide-48, nonapeptide-1, and carnosine can produce synergistic effects in whitening, anti-aging, and anti-oxidation; while the combined use of acetyl hexapeptide-1, palmitoyl pentapeptide-4, and polyamino acid polysaccharide condensates and gold has been applied in firming and anti-wrinkle products. Regarding delivery systems, carriers such as polymer network structured hydrogel spheres and liposomes are used to overcome the skin barrier and improve the transdermal absorption rate of peptides. Current technologies focus on modifying short peptides and polypeptides to enhance their whitening, anti-aging, and antioxidant capabilities. Traditional single proteins have limited functions, and natural proteins suffer from poor stability, difficulty in maintaining activity, and high production costs, making it difficult to meet the demand for efficient, safe, and stable raw materials in cosmetics and medical devices.

[0004] Therefore, there is an urgent need in this field for a fusion protein with whitening, moisturizing, repairing, firming and anti-wrinkle effects. Summary of the Invention

[0005] The purpose of this invention is to provide a fusion protein with whitening, moisturizing, repairing, firming and anti-wrinkle effects and its preparation method.

[0006] Another objective of this invention is to provide a cosmetic product with whitening, moisturizing, repairing, firming, and anti-wrinkle effects.

[0007] A first aspect of the present invention provides a fusion protein comprising the following elements fused together: (a) Annexin A5; and (b) Type III collagen.

[0008] In another preferred embodiment, the fusion protein further includes a linker peptide that connects annexin A5 and type III collagen.

[0009] In another preferred embodiment, the fusion protein has a structure as shown in formula (I): XLY (I) In the formula, X represents annexin A5; L represents no or no connector; Y represents type III collagen; "-" indicates either no peptide or a linked peptide.

[0010] In another preferred embodiment, the connector includes a flexible connector and a rigid connector.

[0011] In another preferred embodiment, the linker is a flexible linker; preferably, it is a GS linker composed of glycine and serine.

[0012] In another preferred embodiment, the connector shown includes (GGGGS)2 or (EAAAK).

[0013] In another preferred embodiment, the annexin A5 comprises full-length annexin A5, its active fragment, or its core functional fragment; the type III collagen comprises full-length type III collagen, its active fragment, or its core functional fragment.

[0014] In another preferred embodiment, the annexin A5 includes human annexin A5, and the type III collagen includes human type III collagen.

[0015] In another preferred embodiment, the amino acid sequence of annexin A5 is shown in SEQ ID NO:1, and the amino acid sequence of type III collagen is shown in SEQ ID NO:2.

[0016] In another preferred embodiment, the amino acid sequence of the annexin A5 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1.

[0017] In another preferred embodiment, the amino acid sequence of the type III collagen has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:2.

[0018] In another preferred embodiment, the sequence of the fusion protein is as shown in SEQ ID NO:3, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.

[0019] In a second aspect, the present invention provides a polynucleotide that encodes a fusion protein as described in the first aspect of the present invention.

[0020] In another preferred embodiment, the polynucleotide includes DNA, RNA, cRNA, or a combination thereof.

[0021] In another preferred embodiment, the sequence of the polynucleotide is shown in SEQ ID NO:4.

[0022] In a third aspect, the present invention provides a carrier containing the polynucleotides as described in the second aspect of the present invention.

[0023] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0024] In a fourth aspect, the present invention provides an engineered host cell containing a vector or genome as described in the third aspect of the present invention, wherein the host cell integrates the polynucleotides described in the second aspect of the present invention.

[0025] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, plant cell, or mammalian cell (including human and non-human mammals).

[0026] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0027] In another preferred embodiment, the host cell is a 293 cell or a CHO cell.

[0028] In a fifth aspect, the present invention provides a method for preparing a fusion protein as described in the first aspect, comprising the steps of: (s1) Under suitable conditions, host cells as described in the fourth aspect of the present invention are cultured to obtain a mixture containing the fusion protein as described in the first aspect of the present invention, and (s2) The mixture obtained in step (s1) is purified and / or separated to obtain the fusion protein as described in the first aspect of the present invention.

[0029] The sixth aspect of the present invention is the use of the fusion protein as described in the first aspect of the present invention for the preparation of cosmetics, cosmetic compositions, pharmaceuticals, or pharmaceutical compositions.

[0030] In another preferred embodiment, the cosmetic, cosmetic composition, drug, or drug composition has the following effects: whitening, moisturizing, anti-wrinkle, firming, repairing, or a combination thereof.

[0031] In a seventh aspect, the present invention provides a cosmetic composition comprising (1) a fusion protein as described in the first aspect of the present invention; and (2) a cosmetically acceptable carrier or excipient.

[0032] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0033] Figure 1 The results showed the efficacy of different proteins in inhibiting melanocytes.

[0034] Figure 2 The results showed the efficacy of different proteins in promoting epidermal cell function. Detailed Implementation

[0035] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors have unexpectedly discovered a fusion protein for the first time. This fusion protein comprises a core functional fragment of annexin A5, a core functional fragment of type III collagen, and a flexible peptide. The fusion protein of this invention simultaneously possesses the anti-inflammatory and melanin-inhibiting activities of annexin A5 and the repair-promoting and anti-aging functions of type III collagen, and exhibits excellent stability, biocompatibility, and application performance.

[0036] Specifically, cell experiments were used to verify the promoting effects of the fusion protein on cell adhesion and proliferation, and human efficacy tests were used to verify its anti-wrinkle and firming effects. The comprehensive performance of the fusion protein in whitening (inhibiting melanin formation), moisturizing (increasing skin hydration), and repairing (accelerating wound healing) was tested, demonstrating that it possesses the activities of two proteins simultaneously, superior to the activity of a single protein. Based on these findings, this invention was completed.

[0037] the term To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0038] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0039] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0040] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0041] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0042] Annexin A5 Human annexin A5 is a calcium-dependent phospholipid-binding protein with biological functions such as anticoagulation, anti-inflammation, and inhibition of apoptosis. Its core functional domain (such as the amino acid sequence QVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAV, SEQ ID NO:1) can exert antithrombotic and anti-inflammatory effects by binding to phosphatidylserine (PS) exposed cell membranes. However, natural annexin A5 suffers from poor stability, a short half-life, and is difficult to directly apply in cosmetics or medical devices.

[0043] Type III collagen Human type III collagen is an important structural protein in tissues such as skin and blood vessels. Its core functional fragment (such as the amino acid sequence NGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMG, SEQ ID NO:2) has the function of promoting cell adhesion, proliferation, migration and tissue repair.

[0044] The fusion protein of the present invention As used herein, the terms "fusion protein of the present invention" and "recombinant fusion protein of the present invention" are used interchangeably and both refer to the fusion protein described in the first aspect of the present invention.

[0045] The fusion protein of the present invention comprises the following protein elements: (a) Annexin A5; and (b) Type III collagen.

[0046] The fusion protein of the present invention includes human annexin A5 as annexin A5 and human type III collagen as type III collagen.

[0047] The fusion protein of this invention has the activity of annexin A5 and the function of human type III collagen. It can be used to prepare cosmetics with whitening, moisturizing, repairing, firming and anti-wrinkle effects, as well as medical devices for skin damage repair and postoperative care.

[0048] In one exemplary embodiment, the amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO:3.

[0049] The fusion proteins of the present invention also include variations of the aforementioned fusion proteins. These variations include (but are not limited to): deletions, insertions, and / or substitutions of 1-5 amino acids (typically 1-3, more preferably 1); additions or deletions of one or more amino acids (typically up to 5, preferably up to 3, more preferably up to 1) at the C-terminus and / or N-terminus; or additions of amino acid fragments with small side chains as linkers (such as glycine, serine, etc.) at the N-terminus or C-terminus of the protein. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding or deleting one or more amino acids at the C-terminus and / or N-terminus generally does not alter the structure and function of the protein. Furthermore, the term also includes the polypeptides of the present invention in monomeric and multimeric forms. The term also includes linear and non-linear polypeptides (such as cyclic peptides).

[0050] The present invention also includes active fragments, derivatives, and analogs of the above-described fusion protein. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the function or activity of the fusion protein of the present invention.

[0051] The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing a polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, for example, polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6His). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0052] A preferred class of active derivatives refers to polypeptides formed by replacing up to five, more preferably up to three, and even more preferably up to one amino acid with an amino acid of similar or analogous properties, compared to the amino acid sequence of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0053] Table A This invention also provides analogs of the fusion protein of this invention. These analogs may differ from the peptides of this invention in terms of amino acid sequence, or in the form of modifications that do not affect the sequence, or both. Analogs also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the peptides of this invention are not limited to the representative peptides exemplified above.

[0054] Furthermore, the fusion protein of the present invention can be modified. Modifications (generally without altering the primary structure) include chemically derived forms of the peptide, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during peptide synthesis and processing or further processing steps. This modification can be accomplished by exposing the peptide to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include peptides modified to improve their resistance to proteolytic hydrolysis or optimize their solubility.

[0055] The term "polynucleotide of the present invention" can refer to a polynucleotide that encodes the fusion protein of the present invention, or it can refer to a polynucleotide that also includes additional coding and / or non-coding sequences.

[0056] The present invention also relates to variants of the aforementioned polynucleotides that encode fragments, analogs, and derivatives of polypeptides or fusion proteins having the same amino acid sequence as those of the present invention. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the fusion protein it encodes.

[0057] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%.

[0058] The fusion proteins and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, purified to homogenization.

[0059] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0060] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0061] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0062] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0063] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0064] expression carrier The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or the coding sequences of the fusion proteins of the present invention, and methods for generating the polypeptides of the present invention via recombinant technology.

[0065] In this invention, the polynucleotide sequence encoding the fusion protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0066] In the fusion protein preparation method of the present invention, any suitable vector can be used, selected from pET, pDR1, pcDNA3.1(+), pcDNA3.1 / ZEO(+), pDHFR, and the expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0067] Both eukaryotic and prokaryotic host cells can be used for the expression of the fusion protein of the present invention. The eukaryotic host cells are preferably mammalian or insect host cell culture systems, preferably COS, CHO, NSO, sf9 and sf21 cells; the prokaryotic host cells are preferably one of DH5a, BL21(DE3) and TG1.

[0068] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0069] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0070] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0071] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces, Salmonella typhimurium bacterial cells, fungal cells such as yeast, and plant cells (such as ginseng cells).

[0072] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0073] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0074] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0075] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0076] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0077] Affinity chromatography can be used to separate and purify a class of fusion proteins disclosed in this invention. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the fusion proteins bound to the affinity column.

[0078] Using the above method, the fusion protein can be purified into a basically homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis.

[0079] cosmetic compositions The present invention also provides cosmetic compositions containing the fusion protein of the present invention. The cosmetic compositions of the present invention comprise (a) the fusion protein of the present invention; and (b) a cosmetically acceptable carrier or excipient.

[0080] The fusion protein of the present invention can be prepared into various cosmetic compositions, such as emulsions, liquids, ointments, creams, pastes, cakes, powders, etc., using conventional methods.

[0081] Within the scope that does not impair the effects of the present invention, other ingredients commonly used in cosmetics may be added to the cosmetics of the present invention, such as film-forming agents, oil-soluble gelling agents, organically modified clay minerals, resins, moisturizers, preservatives, antibacterial agents, fragrances, salts, antioxidants, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell activators, skin roughness improvers, blood circulation promoters, skin astringents, anti-seborrheic agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, etc.

[0082] Oil-soluble gelling agents are selected from metal soaps such as aluminum stearate, magnesium stearate, and zinc myristate; amino acid derivatives such as N-lauroyl-L-glutamic acid and α,γ-di-n-butylamine; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, and cyclodextrin 2-ethylhexanoate palmitate; sucrose fatty acid esters such as sucrose palmitate and sucrose stearate; benzylidene derivatives of sorbitol such as benzylidene sorbitol and dibenzylidene sorbitol; and organically modified clay minerals such as dimethylbenzyl dodecylammonium montmorillonite clay and dimethyl octadecylammonium montmorillonite clay. One, two, or more can be used as needed.

[0083] Moisturizers include: glycerin, sorbitol, propylene glycol, dipropylene glycol, 1,3-butanediol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside, etc.

[0084] Antibacterial and preservative agents include: alkyl p-hydroxybenzoate, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc. Antibacterial agents include: benzoic acid, salicylic acid, carbolic acid, sorbic acid, alkyl p-hydroxybenzoate, p-chloro-m-cresol, hexachlorophenol, benzalkonium chloride, chlorhexidine chloride, trichloro-N-carbonylaniline, triclosan, photosensitizer, phenoxyethanol, etc.

[0085] Antioxidants include: tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, phytic acid, etc.; pH adjusters include: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate, etc.; chelating agents include: alanine, sodium ethylenediaminetetraacetate, sodium polyphosphate, sodium metaphosphate, phosphoric acid, etc.; cooling agents include: L-menthol, camphor, etc.; anti-inflammatory agents include: allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, azulene, etc.

[0086] Skin-beautifying ingredients include: whitening agents such as placental extract, arbutin, glutathione, and saxifrage extract; cell activators such as royal jelly, photosensitizers, cholesterol derivatives, and calf blood extract; skin roughness improvers; blood circulation promoters such as valeramide nonanoate, benzyl nicotinate, β-butoxyethyl nicotinate, capsaicin, gingerone, cantharides tincture, ichthammol, caffeine, tannic acid, α-borneol, nicotinic acid tocopherol, hexanicotinic acid inositol ester, cyclic mandelic acid ester, cinnarizine, tolazoline, acetylcholine, verapamil, senna extract, and γ-oryzanol; skin astringents such as zinc oxide and tannic acid; anti-seborrheic agents such as sulfur; and vitamins such as vitamin A oil, rosin oil, rosin acetate oil, and rosin palmitic acid oil; riboflavin, riboflavin butyrate, and flavin adenine nucleotide. Vitamins include: B2; B6 (such as pyridoxine hydrochloride, pyridoxine dicaprylate, and pyridoxine tripalmitate); B12 and its derivatives; B15 and its derivatives; L-ascorbic acid, L-ascorbic acid dipalmitate, L-ascorbic acid-2-sulfate sodium, and L-ascorbic acid phosphate dipotassium; D (such as ergocalciferol and cholecalciferol); E (such as α-tocopherol, β-tocopherol, γ-tocopherol, dl-α-tocopherol acetate, dl-α-tocopherol niacin, and dl-α-tocopherol succinate); H; P; niacin derivatives (such as niacin, benzyl nicotinate, and nicotinamide); pantothenic acid derivatives (such as calcium pantothenate, D-panthenol, pantothenicotinyl ethyl ether, and acetylpantothenicotinyl ethyl ether); and biotin.

[0087] Amino acids include glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine, tryptophan, etc. Nucleic acids include deoxyribonucleic acid, etc. Hormones include estradiol, vinyl estradiol, etc.

[0088] Preferred examples of cosmetics of the present invention include: skin care cosmetics, color cosmetics, and UV protection cosmetics. Examples include basic cosmetics such as lotions, creams, serums, sunscreens, mask materials, facial cleansers, and solutions; and color cosmetics such as foundations, face powders, and blushes.

[0089] There are no particular restrictions on the form of the product; it can be liquid, emulsion, cream, solid, paste, gel, powder, multi-layered, mousse, spray, etc.

[0090] The present invention also provides a skin care method comprising the steps of: applying the fusion protein of the present invention, or a cosmetic or care product containing the fusion protein of the present invention, to an individual in need.

[0091] In another preferred embodiment, the method is an anti-aging, whitening, moisturizing, firming, or skin repair method.

[0092] The main advantages of this invention include: 1. The fusion protein of the present invention possesses both the anti-inflammatory and melanin-inhibiting activities of annexin A5 and the repair-promoting and anti-aging functions of type III collagen, thus meeting the demand of cosmetics and medical devices for multi-functional raw materials.

[0093] 2. The fusion protein of the present invention optimizes the protein structure through flexible peptide design, thereby improving its thermal stability, pH stability and storage stability.

[0094] 3. The fusion protein of the present invention has no immunogenicity risk, good biocompatibility, and can replace natural collagen to reduce the risk of allergies.

[0095] 4. The fusion protein of the present invention can be mass-produced through genetic engineering methods, thereby reducing costs and improving batch-to-batch consistency.

[0096] 5. The fusion protein of the present invention can be added as an active ingredient to products such as solutions, masks, and lotions to achieve multiple effects such as whitening, moisturizing, anti-wrinkle, and repair; it can also be used in skin repair dressings, anti-scar gels, injectable fillers, etc. to promote tissue regeneration and reduce inflammatory response.

[0097] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0098] Example 1 Construction of recombinant fusion protein Escherichia coli amino acid sequence: MGDKSK-QVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAV-GGGGS-GGGGS-EAAAK-NGQGPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMG(SEQ ID NO:3) After codon optimization: ATGGGTGATAAAAGCAAACAGGTGGAACAGGATGCGCAGGCGCTGTTTCAGGCGGGTGAACTGAAATGGGGTACCGATGAAGAAAAATTTATTACCATTTTTGGTACCCGTAGCGTGAGCCATCTGCGTAAAGTGTTTGATAAATATATGACCATTAGCGGTTTTCAGATTGAAGAAACCATTGATCGTGAAACCAGCGGTAACCTGGAACAGCTGCTGCTGGCGGTGGGTGGTGGTGGTAGCGGTGGTGGTGGTAGCGAAGCGGCGGCGAAAAACGGTCAGGGTCCGCAGGGTCCGAAAGGTGATCCGGGTCCGCCGGGTATTCCGGGTCGTAACGGTGATCCGGGTATTCCGGGTCAGCCGGGTAGCCCGGGTAGCCCGGGTCCGCCGGGTATTTGTGAAAGCTGTCCGACCGGTCCGCAGAACTATAGCCCGCAGTATGATAGCTATGATGTGAAAAGCGGTGTGGCGGTGGGTGGTCTGGCGGGTTATCCGGGTCCGGCGGGTCCGCCGGGTCCGCCGGGTCCGCCGGGTACCAGCGGTCATCCGGGTAGCCCGGGTAGCCCGGGTTATCAGGGTCCGCCGGGTGAACCGGGTCAGGCGGGTCCGAGCGGTCCGCCGGGTCCGCCGGGTGCGATTGGTCCGAGCGGTCCGGCGGGTAAAGATGGTGAAAGCGGTCGTCCGGGTCGTCCGGGTGAACGTGGTCTGCCGGGTCCGCCGGGTATTAAAGGTCCGGCGGGTATTCCGGGTTTTCCGGGTATGAAAGGTCATCGTGGTTTTGATGGTCGTAACGGTGAAAAAGGTGAAACCGGTGCGCCGGGTCTGAAAGGTGAAAACGGTCTGCCGGGTGAAAACGGTGCGCCGGGTCCGATGGGT (SEQ ID NO:4) Expression strain: BL21(DE3) pLysS Expression vector: pET-28a(+) Restriction enzyme sites: Nco I and Xho I restriction enzyme sites Other requirements: 5' - add His label, 3' - add terminator The gene is cloned into an expression vector (such as the pET-28a(+) series) to construct a recombinant plasmid. This plasmid is then transformed into *E. coli*, yeast, or mammalian cells for expression. The target protein is purified using methods such as affinity chromatography or ion exchange chromatography.

[0099] Example 2: Detection of recombinant fusion protein cell viability 2.1 Experimental Verification of Efficacy in Inhibiting Melanocytes 1. Selection of cell models B16F10 mouse melanoma cells (derived from ATCC).

[0100] 2. Grouping of solution experiments Negative control group (placebo): DMEM medium containing 5% FBS + flexible peptide GGGGSGGGGSEAAAK (SEQ ID NO:5) (50 μg / ml) Single proteome: A5 core functional fragment (SEQ ID NO:1) solution (50 μg / ml) Type III collagen core functional fragment (SEQ ID NO:2) solution (50 μg / ml) Fusion protein group: Fusion protein (SEQ ID NO:3) solution group (50 μg / ml) 3. Core detection indicators and methods for solutions (1) Determination of melanin content Operating steps: Cells were seeded in 6-well plates (2 × 10⁻⁶ cells per well). 5 (each well contains 1 sample), and after adhesion, the protein from each group is added and treated for 48-72 hours; Cells were collected by trypsin digestion, washed twice with PBS solution, and centrifuged at 1000 rpm for 5 min. Add 100 μL of 1M NaOH (containing 10% DMSO) to the precipitate and incubate in a water bath at 80°C for 30 min to dissolve the melanin; The absorbance (OD value) at 475 nm was measured using an ELISA reader. A standard curve was plotted using a standard (melanin standard) to calculate the melanin content of each group.

[0101] 4. Results Based on the results of melanin formation inhibition, neither the type III collagen core domain nor the flexible linker peptide exhibited melanin formation inhibition efficacy. Both the fusion protein and the A5 protein core domain retained melanin formation inhibition efficacy, with the fusion protein demonstrating superior ability to inhibit melanin formation compared to the A5 protein core domain alone. Figure 1 ).

[0102] 2.2 Experiment to verify the efficacy of promoting epidermal cell function 1. Selection of cell models HaCaT is an immortalized human keratinocyte derived from ATCC.

[0103] 2. Experimental Grouping Negative control group (placebo): DMEM medium containing 5% FBS + flexible peptide solution GGGGSGGGGSEAAAK (50 μg / ml) Single proteome: A5 core functional fragment (SEQ ID NO:1) solution (50 μg / ml) Type III collagen core functional fragment (SEQ ID NO:2) solution (50 μg / ml) Fusion protein group: Fusion protein (SEQ ID NO:3) (50 μg / ml) 3. Detection of epidermal cell proliferation capacity Operating steps: With 3×10 per hole 3 Cells were seeded at a density of 1,000 cells in 96-well plates. After cell adhesion, the cells were co-cultured with each group of proteins, and cell proliferation was assessed on day 3. After discarding the culture medium, add 100 μL of CCK-8 reagent diluted 10 times to each well and incubate at 37°C for 2 hours; The OD value of each well was measured at a wavelength of 450 nm using an ELISA reader. Statistical analysis: Relative OD value = OD value of experimental group - OD value of blank group.

[0104] 4. Results Based on the epidermal cell proliferation results, neither the A5 protein core domain nor the flexible linker peptide exhibited cell proliferation-promoting effects. Both the fusion protein and the type III collagen core domain retained their epidermal cell proliferation-promoting effects, with the fusion protein demonstrating superior proliferation-promoting ability compared to the type III collagen core domain alone. Figure 2 ).

[0105] Example 3: Human Efficacy Evaluation Experiment of Recombinant Fusion Protein This embodiment aims to evaluate the moisturizing, repairing, firming, and anti-wrinkle effects of the recombinant fusion protein on the skin, as well as its effect on improving skin tone and pigmentation, through human efficacy evaluation. By using the test product on subjects, combined with instrumental testing and subjective assessment, the moisturizing, repairing, firming, and anti-wrinkle effects of the solution, as well as its effect on improving skin tone and pigmentation, are comprehensively evaluated.

[0106] 3.1 Test Sample: The purified protein from Example 1 was prepared into a 0.1 mg / ml solution (the fusion protein was dissolved in 0.9% sodium chloride, with a final concentration of 0.1 mg / ml). 3.2 References for the Solution: 《T / CAB 0152-2022: Test Methods for Seven Efficacy Items of Cosmetics (Anti-wrinkle, Firming, Moisturizing, Oil Control, Repairing, Nourishing, and Soothing) T / GDCA 009-2022: Human Evaluation Methods for Repairing Efficacy of Cosmetics》; T / TDCA 003-2021: Test Method for Firming Efficacy of Cosmetics; Liu Weiyi, Zhou Lin, Zhao Hua. Cosmetic Efficacy Evaluation (XIII) - Consumer Use Test [J] Daily Chemical Industry, 2021, 51(06): 485-490.

[0107] 3.3 Purpose of the test: To comprehensively evaluate the product’s moisturizing, repairing, firming, anti-wrinkle effects and its effect on improving skin color and pigmentation by having subjects use the test product and by using instrument detection and subjective evaluation.

[0108] 3.4 Detection Methods: Thirty-three healthy Chinese male / female subjects were selected, characterized by dry facial skin lacking moisture, poor barrier function, loose and inelastic skin, visible pigmentation, pronounced facial texture, fine lines or wrinkles, and under-eye wrinkles conforming to the Skinning Atlas scale (grades 2-5). Subjects numbered RD0020 and RD0029 withdrew from the trial due to personal reasons, leaving 31 subjects in total (5 males and 26 females), aged 27 to 58 years, with a mean age of 44.74 ± 1.66 years. A before-and-after control method was used to measure the subjects' stratum corneum moisture content, transepidermal water loss, skin elasticity, skin firmness, and melanin index. Subjective assessments were conducted, and full-face photographs were taken. The results were compared using statistical tests to determine if there were statistically significant differences.

[0109] 3.5 Detection Site (1) Skin stratum corneum moisture content - cheeks (2) Transepidermal water loss value of skin - cheek (3) Skin elasticity R2—cheek (4) Skin firmness F4 - cheeks (5) Skin melanin index (MI) – indicating the location of pigmentation spots (6) VISIA-CR under-eye wrinkle area - full face photo analysis of under-eye area (7) VISIA-CR under-eye wrinkle area percentage - full-face photo analysis of under-eye area (8) VISIA-CR Skin Color L* Value—Full Face Photo Analysis of Cheeks (9) VISIA-CR Skin Color ITA° Value—Full Face Photo Analysis of Cheeks (10) VISIA-CR Skin Gloss Parameters - Full Face Photo Analysis of Cheeks (11) VISIA-CR mean light density of pigment spots - full face photo analysis of cheeks (12) VISIA-CR pigmentation area ratio - full face photo analysis of cheeks Table 1. Detection Indicators and Specific Detection Methods Note: D0 is day 0 of the test, D14 is day 14 of the test, and so on.

[0110] 3.6 Statistical Methods: SPSS was used for statistical analysis. If the values ​​were normally distributed, the T-test was used; if the values ​​were not normally distributed, the rank-sum test was used. All statistical methods employed two-tailed tests with a significance level of α = 0.05.

[0111] 3.7 Test Results 3.7.1 Sample Completion Status: 33 participants were enrolled. Participants numbered RD0020 and RD0029 withdrew from the trial due to personal reasons, leaving 31 participants in total. These included 5 males and 26 females, aged 27 to 58 years, with a mean age of 44.74 ± 1.66 years. Fusion Protein Usage: Apply an appropriate amount of the fusion protein solution to the face and gently massage until absorbed. Use twice daily, morning and evening.

[0112] 3.7.2 Safety Evaluation Each subject used the test product as required within the specified time. Through questioning, examination, and recording, any skin reactions or systemic adverse reactions experienced by the subjects during the trial were documented, including the manifestation, time of occurrence, treatment measures, and outcome of the adverse events. The relationship between the adverse events and the test product used was also assessed. See Table 2 below for details: Table 2. Skin reactions in human trials As shown in Table 2, no adverse skin reactions were observed in any of the 31 subjects throughout the trial.

[0113] 3.7.3 Summary of Human Efficacy Evaluation Test Results After 14 and 28 days of continuous use of the recombinant fusion protein, the skin stratum corneum moisture content, transepidermal water loss, skin elasticity (R2), skin firmness (F4), VISIA-CR under-eye wrinkle area, and VISIA-CR under-eye wrinkle area percentage were significantly improved compared with the baseline values. Therefore, it is believed that the fusion protein has moisturizing, repairing, anti-wrinkle, and firming effects.

[0114] After continuous use of the experimental product, the recombinant fusion protein, for 14 and 28 days, the skin melanin index (MI), VISIA-CR skin color L* value, VISIA-CR skin color ITA° value, VISIA-CR pigmentation area ratio, and VISIA-CR skin gloss parameters showed significant improvements compared to baseline values. Therefore, it is believed that the recombinant fusion protein has the effect of improving skin color and pigmentation.

[0115] Table 3. Results of human efficacy tests of recombinant fusion protein All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises the following elements fused together: (a) annexin A5; and (b) collagen type III.

2. The fusion protein of claim 1, wherein, The fusion protein has a structure as shown in formula (I): X-L-Y (I) In the formula, X is annexin A5; L is nothing or a linker; Y is collagen type III; Each of the “-” is independently nothing or a linking peptide.

3. The fusion protein of claim 1, wherein, The annexin A5 includes full-length annexin A5, an active fragment thereof, or a core functional fragment thereof; the collagen type III includes full-length collagen type III, an active fragment thereof, or a core functional fragment thereof.

4. The fusion protein of claim 1, wherein, The amino acid sequence of the annexin A5 is shown in SEQ ID NO: 1, and the amino acid sequence of the collagen type III is shown in SEQ ID NO:

2.

5. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-4. The polynucleotide encodes the fusion protein as claimed in claim 1.

6. A vector, characterized in that, The vector contains the polynucleotide as claimed in claim 5.

7. An engineered host cell, characterized in that, The host cell contains the vector as claimed in claim 6 or the polynucleotide as claimed in claim 5 integrated in the genome.

8. A method of preparing the fusion protein of claim 1, wherein, The method comprises the steps of: (s1) culturing the host cell as claimed in claim 7 under suitable conditions, thereby obtaining a mixture containing the fusion protein as claimed in claim 1, and (s2) purifying and / or isolating the mixture obtained in step (s1), thereby obtaining the fusion protein as claimed in claim 1.

9. Use of the fusion protein according to claim 1, characterized in that, For preparing a cosmetic, a cosmetic composition, a drug, or a pharmaceutical composition.

10. A cosmetic composition characterized in that, Comprising (1) the fusion protein as claimed in claim 1; and (2) a cosmetically acceptable carrier or excipient.

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