Use of recombinant human elastin peptide with Anti-aging effects and composition thereof

Abstract

Relating to the field of biotechnology, disclosed are a recombinant human elastin peptide with anti-aging effects and a use thereof. The recombinant human elastin peptide comprises an amino acid sequence shown in SEQ ID NO:1 or a fragment of the amino acid sequence shown in SEQ ID NO:1, the fragment comprises 30% or more of SEQ ID NO:1, or a mutant of the amino acid sequence shown in SEQ ID NO:1 or the fragment of the amino acid sequence shown in SEQ ID NO:1. A disclosed method for producing elastin peptides by using a yeast expression system enables the targeted synthesis of highly effective protein fragments with 100% homology to human proteins, while achieving low costs and facilitating large-scale production, avoiding animal-derived sources and viral risks. This makes the recombinant human elastin peptide safe for use as an anti-aging and anti-photoaging active ingredient in cosmetics, while also supporting a use thereof in the field of biomedical materials.

Description

Application of a recombinant human elastin peptide with anti-aging effect and its composition

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application No. 202410387107.X, filed on April 1, 2024, entitled “A recombinant human elastin peptide with anti-aging efficacy and its application in a composition thereof,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biotechnology, and in particular to a recombinant human elastin peptide with anti-aging efficacy and application thereof. Background Art

[0004] Skin aging refers to the gradual loss of skin elasticity and firmness with age. It is caused by a variety of factors, including natural aging, environmental factors, lifestyle, and genetics. With the improvement of people's living standards, skin aging has received widespread attention. The main characteristics of aging skin are epidermal thinning, flattening of the dermal-epidermal junction, and degradation of the dermal extracellular matrix. Elastin, the second largest protein in the extracellular matrix after collagen, plays a crucial role in skin aging. Its degradation and destruction can cause symptoms such as decreased skin elasticity, sagging, and wrinkles.

[0005] The primary source of elastin is currently animal tissue extraction. This involves collecting elastin-rich tissues or biological samples, such as blood vessels, skin, and lung tissue. After pulverizing the tissue, non-elastin components, such as collagen, are removed using chemical or high-temperature treatment. The elastin is then mixed with a solvent and dissolved and extracted under controlled temperatures, releasing the elastin into solution. The elastin is then filtered, precipitated, washed, and purified to yield a high-purity elastin. The extracted elastin maintains a molecular weight and structure that closely resembles the native protein. However, immunogenicity, viral potential, and the uniqueness of the extracted protein sequence during the extraction process pose challenges to its application in cosmetics and medical applications. In recent years, a number of elastin synthesized using recombinant genetic techniques have emerged. For example, patent number CN202110357328.9 utilizes Saccharomyces cerevisiae to synthesize a repeating sequence of the key elastin hexapeptide GVGVAP. This elastin fragment exhibits excellent biocompatibility, antioxidant properties, and reducibility. For example, CN202211035433.1 describes a method for synthesizing an elastin (VPTGIG) 25-repeat sequence using Escherichia coli. This method simplifies purification and provides a new type of drug carrier. CN202010611388.4 discloses an elastin repeat sequence (VAPGVG) 3S synthesized by Escherichia coli. This fragment, which is repeated 3-7 times in tandem, has been shown to have high superoxide radical scavenging activity.

[0006] However, the existing technology still has technical problems such as high cost investment and being unfavorable for large-scale production. There is an urgent need for the application of a recombinant human elastin peptide with anti-aging effect and its composition to solve such problems.

[0007] Summary of the Invention

[0008] The present invention provides a recombinant human elastin peptide with anti-aging efficacy and application of a composition thereof to solve the above-mentioned problems in the prior art.

[0009] The present invention discloses a recombinant human elastin peptide with anti-aging efficacy, wherein the recombinant human elastin peptide is selected from the following a) to c): a) comprising the amino acid sequence shown in SEQ ID NO: 1; b) comprising a fragment of the amino acid sequence shown in SEQ ID NO: 1, wherein the fragment comprises 30% or more of SEQ ID NO: 1; and c) mutants of the above a) and b).

[0010] In a preferred technical solution, the recombinant human elastin peptide comprises the amino acid sequence shown in SEQ ID NO: 21 or a variant sequence obtained by inserting, substituting or deleting one or more amino acids in SEQ ID NO: 21.

[0011] In a preferred technical solution, the variant sequence has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 21; preferably, the variant retains anti-aging efficacy equivalent to SEQ ID NO: 21.

[0012] In a preferred technical solution, the anti-aging effect is selected from one or more of the following: 1) promoting the expression of endogenous elastin; 2) promoting the expression of collagen IV and / or VII genes in the dermal-epidermal junction layer; 3) protecting collagen I and the like from being destroyed by ultraviolet rays; 4) increasing or maintaining the water content of the skin; 5) improving skin gloss; 6) reducing crow's feet; 7) increasing the L value of the skin; 8) increasing skin density; 9) improving skin elasticity or firmness.

[0013] In a preferred technical solution, the recombinant human elastin peptide consists of the amino acid sequence shown in SEQ ID NO: 21.

[0014] In a preferred technical solution, the N-terminus and / or C-terminus of the recombinant human elastin peptide sequence comprises a fusion protein sequence.

[0015] In a preferred technical solution, the fusion protein sequence is a protein tag.

[0016] In a preferred technical solution, the protein tag is one of a His tag, a glutathione sulfhydryltransferase tag, a maltose binding protein tag, a SUMO tag, a NusA tag, a TrxA tag and a DsbA tag.

[0017] As a preferred technical solution, the recombinant human elastin peptide is expressed by a host; the host is one of bacteria, fungi and eukaryotic cells.

[0018] In a preferred technical solution, the bacteria is one of Escherichia coli, Bacillus subtilis, and Rhodococcus erythropolis; the fungus is one of Pichia pastoris, Saccharomyces cerevisiae, and Aspergillus oryzae; and the eukaryotic cell is one of insect cells, CHO cells, mouse cells, and human cells.

[0019] In a preferred technical solution, the recombinant human elastin peptide is expressed in host cells.

[0020] In a preferred technical solution, the recombinant human elastin peptide is exogenously expressed by host cells.

[0021] In a preferred embodiment, the recombinant human elastin peptide is expressed exocytically, and its precursor protein contains a secretion signal peptide sequence. A secretion signal peptide is a short peptide that directs the newly synthesized protein into the secretory pathway. During extracellular secretion of the mature protein, the secretion signal peptide is removed by a signal peptidase.

[0022] In a preferred technical solution, the recombinant human elastin peptide is exogenously expressed by either Pichia pastoris or Saccharomyces cerevisiae.

[0023] In a preferred technical solution, the precursor protein of the recombinant human elastin peptide comprises a signal peptide sequence of Saccharomyces cerevisiae mating factor α, and the amino acid sequence of the signal peptide of the mating factor α is shown in SEQ ID NO: 2.

[0024] In a preferred technical solution, the gene encoding the recombinant human elastin peptide includes a fragment encoding the elastin peptide in the cDNA sequence of the human elastin gene and an artificially synthesized gene.

[0025] In a preferred technical solution, the recombinant human elastin peptide is expressed exogenously, and its gene encodes the precursor of the recombinant human elastin peptide, which is manifested by its gene including a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding the recombinant human elastin peptide.

[0026] In a preferred embodiment, the gene sequence encoding the recombinant human elastin peptide is a synthetic gene sequence that has undergone codon optimization. Codon optimization is a method of redesigning a gene sequence by avoiding rare codons, utilizing preferred codons, simplifying the secondary structure of mRNA, optimizing repetitive sequences, and adjusting GC content to improve translation efficiency and thereby increase protein expression levels.

[0027] In a preferred technical solution, the artificial synthetic gene encoding the elastin peptide is synthesized by codon-optimizing the nucleotide sequences encoding the signal peptide and the elastin peptide.

[0028] In a preferred technical solution, the artificially synthesized gene encoding the elastin peptide is synthesized by codon-optimizing the nucleotide sequence encoding the signal peptide, the elastin peptide and the C-terminal His tag.

[0029] In a preferred technical solution, the nucleotide sequence of the gene encoding the artificial synthetic elastin peptide is shown as SEQ ID NO: 3 or 31.

[0030] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is expressed via a promoter, which includes an inducible promoter and a constitutive promoter.

[0031] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is expressed via an inducible promoter.

[0032] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is expressed by a constitutive promoter.

[0033] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is introduced into the host cell.

[0034] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is located on a movable element. In a preferred technical solution, the movable element is a plasmid.

[0035] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is integrated into the host genome.

[0036] The present invention also discloses an isolated nucleic acid encoding the recombinant human elastin peptide of the present invention.

[0037] In a preferred embodiment, the isolated nucleic acid is codon-optimized.

[0038] In a preferred embodiment, the isolated nucleic acid is codon-optimized for Escherichia coli or yeast.

[0039] In a preferred technical solution, the isolated nucleic acid is an artificially synthesized gene.

[0040] In a preferred technical solution, the isolated nucleic acid encodes the precursor of the recombinant human elastin peptide, and its gene includes a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding a recombinant human elastin peptide.

[0041] In a preferred technical solution, the isolated nucleic acid encodes the precursor of the recombinant human elastin peptide, and its gene includes a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a recombinant human elastin peptide, and a nucleotide sequence encoding a C-terminal His tag.

[0042] In a preferred technical solution, the isolated nucleic acid is synthesized by codon-optimizing the nucleotide sequence encoding the signal peptide, the elastin peptide and the C-terminal His tag.

[0043] In a preferred technical solution, the isolated nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 3 or 31.

[0044] The present invention also discloses a vector comprising the isolated nucleic acid of the present invention.

[0045] In a preferred technical solution, the vector is a eukaryotic vector or a prokaryotic vector.

[0046] The present invention also discloses a host cell comprising the isolated nucleic acid of the present invention or the vector of the present invention.

[0047] In a preferred technical solution, the host cell is a bacterial, fungal or eukaryotic cell.

[0048] In a preferred technical solution, the bacterial host is one of Escherichia coli, Bacillus subtilis and Rhodococcus erythropolis.

[0049] In a preferred technical solution, the fungal host is one of Pichia pastoris, Saccharomyces cerevisiae and Aspergillus oryzae.

[0050] In a preferred technical solution, the eukaryotic cell is one of an insect cell, a CHO cell, a mouse cell and a human cell.

[0051] The present invention also discloses a method for preparing a recombinant human elastin peptide with anti-aging efficacy from a fermentation culture, comprising the following steps:

[0052] 1) fermenting a host cell containing a gene encoding a recombinant human elastin peptide or a precursor protein thereof;

[0053] 2) Protein purification: obtain recombinant human elastin peptide from the fermentation product.

[0054] For fermentation products expressing intracellularly, recombinant human elastin peptides were prepared from cell lysates; for fermentation products expressing exocrinely, recombinant human elastin peptides were prepared from fermentation supernatants.

[0055] Protein purification can be prepared using conventional methods in the art, such as the method described by Wingfield in “Overview of the purification of recombinant proteins” (Curr Protoc Protein Sci 2015 80:6.1.1-6.1.35.doi:10.1002 / 0471140864.ps0601s80.).

[0056] In a preferred technical solution, the protein is purified by the following method: for fermentation products expressed intracellularly, the fermentation products are lysed, the lysate is centrifuged in a low-temperature centrifuge, and the supernatant is retained; for fermentation products expressed exogenously, the fermentation products are centrifuged in a low-temperature centrifuge, and the supernatant is retained;

[0057] The obtained supernatant was filtered through a 0.45 μm filter membrane, and then the protein was purified through an affinity chromatography column to obtain a recombinant human elastin peptide with anti-aging effect.

[0058] As a preferred technical solution, the affinity chromatography column protein purification steps are as follows:

[0059] S1, flow the sample through the Ni column at a rate of 1 ml / min;

[0060] S2, equilibrate the column with Buffer A;

[0061] S3, elution with 20 mM, 300 mM, and 500 mM imidazole;

[0062] S4. The eluted samples were subjected to SDS-PAGE gel analysis to determine whether the target protein was present.

[0063] The present invention also discloses a recombinant human elastin peptide composition with anti-aging efficacy, wherein the recombinant human elastin peptide comprises 0.01% to 5% of the composition, and the rest is a formula agent.

[0064] In the preferred technical solution, the formulation includes the following components in weight percentage: 0.05-1 parts by weight of a chelating agent, 0.1-0.5 parts by weight of a thickener, 3.0-6.0 parts by weight of a polyol, 2-5 parts by weight of vegetable oils and fats, 1-3 parts by weight of a high-melting-point fatty compound, 3-20 parts by weight of a skin moisturizer, 2-10 parts by weight of an emulsifier, 1-3 parts by weight of a preservative, a pH adjustment system to 6.0-7.0, and the remainder is made up to 100 parts by weight with water.

[0065] The present invention also discloses a recombinant human elastin peptide composition with anti-aging efficacy, comprising a recombinant human elastin peptide and excipients; preferably, the recombinant human elastin peptide comprises the amino acid sequence shown in SEQ ID NO: 21; preferably, the composition is an emulsion, an aqueous solution or an aqueous solution.

[0066] In a preferred technical solution, the composition is in the form of an aqueous solution, and the excipients include a solvent, a humectant, a penetration enhancer, and a solubilizing agent. Preferably, the solvent is water; preferably, the humectant includes propylene glycol; preferably, the penetration enhancer includes tetrahydropiperine and / or tridecapeptide-1; and preferably, the solubilizing agent includes PEG-40 hydrogenated castor oil.

[0067] In a preferred technical solution, the dosage form of the composition is an emulsion, and the excipients include one or more selected from solvents, chelating agents, thickeners, emulsifiers, emollients, moisturizers, penetration enhancers, and pH regulators; preferably, the solvent is water; preferably, the chelating agent is disodium EDTA; preferably, the thickener includes one or more of carbomer, xanthan gum, and polyacrylate crosspolymer-6; preferably, the emulsifier is selected from methyl glucose sesquistearate and / or PEG-20 methyl glucoside sesquistearate; preferably, the emollient is selected from polydimethylsiloxane and / or triethylhexanoin; preferably, the penetration enhancer is selected from one or more of phytol, tetrahydropiperine, and tridecapeptide-1; preferably, the moisturizer includes propylene glycol.

[0068] The present invention also discloses the use of a recombinant human elastin peptide composition with anti-aging efficacy in the preparation of skin care products, skin repair dressings, implants, artificial skin, medical devices, and biomaterials.

[0069] Advantages of the present invention:

[0070] 1) This invention utilizes Pichia pastoris to ferment and synthesize a functional fragment of human elastin. This fragment significantly promotes the expression of native elastin and the synthesis of collagen IV and VII genes at the dermal-epidermal junction. UV-resistant experiments have demonstrated that this recombinant human elastin fragment can protect collagen I and other proteins from UV damage. Clinical trials have confirmed that an emulsion containing this recombinant elastin fragment significantly improves dermal density and elasticity, while also significantly improving skin hydration and roughness.

[0071] Transdermal experiments have shown that the human elastin peptide of the present invention also has good transdermal absorption performance.

[0072] 2) The present invention adopts gene recombination technology, accesses the genome of Pichia pastoris, utilizes the expression system of Pichia pastoris, efficiently synthesizes elastin, and then obtains high-purity recombinant elastin by separation and purification.

[0073] This method enables the targeted synthesis of highly effective fragments with 100% homology to human proteins, while also being cost-effective, easy to mass-produce, and avoiding animal sources and viral hazards. This allows the safe use of this recombinant human elastin as an anti-aging and anti-photoaging ingredient in cosmetics, while also supporting its application in the field of biomedical materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG1 is a map of the integration plasmid pELN1 of the recombinant human elastin peptide (SEQ ID NO: 1) of the present invention, wherein “elastin peptide” represents the coding sequence of the recombinant human elastin peptide of SEQ ID NO: 1.

[0075] FIG2 is an electrophoretic diagram of PCR verification of the insertion of the elastin peptide integration plasmid pELN1 into the transformant genome according to an embodiment of the present invention.

[0076] FIG3 is an SDS-PAGE image of the recombinant elastin peptide (SEQ ID NO: 1) after fermentation and purification according to an embodiment of the present invention.

[0077] Figure 4 shows the promoting effect of recombinant elastin on elastin gene expression in fibroblasts. As the concentration of recombinant elastin (ELN) increases, the elastin gene expression effect increases significantly, reaching a high value at 15 ppm. ELN on the abscissa in Figure 4 represents recombinant elastin peptide, and ELN on the ordinate represents elastin synthesized by fibroblasts. NT in Figure 4 indicates no treatment.

[0078] FIG5 is a diagram showing the promoting effect of recombinant elastin on collagen VII gene expression in the present invention, wherein, with the increase in the concentration of recombinant elastin (ELN), the gene expression effect of collagen VII increased significantly, showing concentration dependence; NT in FIG5 represents no treatment.

[0079] FIG6 is a diagram showing the promoting effect of the recombinant elastin of the present invention on the gene expression of collagen IV. As the concentration of recombinant elastin (ELN) increases, the gene expression effect of collagen IV increases significantly, showing concentration dependence. NT in FIG6 indicates no treatment.

[0080] FIG7 is a comparative diagram of UVA irradiation of the present invention, in which the gene expression of collagen I in fibroblasts after UVA irradiation is significantly reduced, while the gene expression of collagen I in fibroblasts treated with recombinant elastin (ELN) is restored, demonstrating that recombinant elastin has significant UVA protection efficacy.

[0081] FIG8 is a comparison chart of skin moisture content after 14 days of use of an emulsion formula containing 60 ppm of recombinant elastin in the present invention. The skin moisture content of the 2% ELN (i.e., the product of Example 3 of the present invention) was twice that of the control group (placebo), and the effect became more significant with increasing use time.

[0082] FIG9 is a comparison chart showing the skin glossiness of the emulsion formula containing 60 ppm recombinant elastin after 14 days of use. The 2% ELN (i.e., the product of Example 3 of the present invention) showed a significant increase in skin glossiness, and with increasing use time, the skin glossiness improved by two times after two months.

[0083] FIG10 is a comparison chart showing crow's feet wrinkles after 14 days of use using an emulsion formula containing 60 ppm of recombinant elastin in the present invention. 2% ELN (i.e., the product of Example 3 of the present invention) significantly reduces crow's feet wrinkles, and the effect becomes more pronounced with increasing usage time.

[0084] FIG11 is a comparison chart of the skin L values ​​after two months of use of an emulsion formula containing 60 ppm of recombinant elastin in the present invention. The L value of the skin treated with 2% ELN (i.e., the product of Example 3 of the present invention) was significantly increased compared to the control group (placebo), demonstrating that the formula has a certain whitening effect.

[0085] Figure 12 is a map of the integration plasmid pELN2 of the recombinant human elastin peptide (SEQ ID NO: 21) of the present invention, wherein "elastin truncated peptide" refers to the coding sequence of the recombinant human elastin peptide of SEQ ID NO: 21.

[0086] FIG13 is an electrophoretic diagram of PCR verification of the insertion of the recombinant human elastin peptide integration plasmid pELN2 into the transformant genome in an embodiment of the present invention.

[0087] FIG14 is an SDS-PAGE image of the fermentation supernatant of the recombinant human elastin peptide (SEQ ID NO: 21) according to an embodiment of the present invention.

[0088] FIG15 is an elution curve of a recombinant human elastin peptide fragment labeled with FITC according to an embodiment of the present invention.

[0089] FIG16 is a standard curve of fluorescence quantification of recombinant human elastin peptide labeled with FITC according to an embodiment of the present invention.

[0090] Figure 17 shows the distribution of FITC-elastin in the skin using CLSM in different formulations, as measured in an embodiment of the present invention. A, recombinant human elastin peptide aqueous solution; B, recombinant human elastin peptide emulsion; C, recombinant human elastin peptide aqueous solution.

[0091] FIG18 is a comparison chart of dermal density of the emulsion formula containing 60 ppm of recombinant elastin peptide according to the present invention after 14 days, 28 days, and 56 days of use (compared with the control group).

[0092] FIG19 is a comparison chart of skin elasticity of the emulsion formula containing 60 ppm of recombinant elastin peptide according to the present invention after 14 days, 28 days, and 56 days of use (compared with the control group).

[0093] FIG20 is a comparison chart of skin firmness of the emulsion formula containing 60 ppm of recombinant elastin peptide according to the present invention after 14 days, 28 days, and 56 days of use (compared with the control group).

[0094] FIG21 is a comparison chart of skin roughness of the emulsion formula containing 60 ppm of recombinant elastin peptide according to the present invention after 14 days, 28 days, and 56 days of use (compared with the control group).

[0095] Detailed Description of the Invention

[0096] In order to make up for the above deficiencies, the present invention provides a recombinant human elastin peptide with anti-aging efficacy and an application of a composition thereof to solve the problems in the above background technology.

[0097] Definition of terms

[0098] As used herein, the term "peptide" or "polypeptide" refers to a polymer of amino acid residues. A "peptide" or "polypeptide" may be modified (e.g., phosphorylated or glycosylated) or unmodified. A "polypeptide" may comprise "conservative substitutions," which, with respect to an amino acid sequence, refer to replacement of an amino acid residue with a different amino acid residue containing a side chain having similar physicochemical properties. For example, conservative substitutions may be made between amino acid residues having hydrophobic side chains, between amino acid residues having neutral hydrophilic side chains, between amino acid residues having aromatic side chains, between amino acid residues having acidic side chains, or between amino acid residues having basic side chains. As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained.

[0099] As used herein, "vector" refers to a DNA molecule that is used in genetic engineering recombinant DNA technology to transfer DNA fragments (target genes) to recipient cells. Vectors can be divided into cloning vectors and expression vectors. Cloning vectors are mainly used to clone and amplify DNA fragments. They mainly include plasmid vectors, phage vectors, phagemid vectors, and viral vectors. In addition to the basic elements of a cloning vector, an expression vector also has the control elements necessary for transcription and translation, such as promoters and terminators.

[0100] As used herein, an "integrating plasmid" refers to a DNA sequence comprising a selectable marker and at least one target gene expression cassette. Prior to being transformed into yeast, the integrating plasmid is linearized, which allows its sequence to be inserted into the yeast genomic region.

[0101] As used herein, the term "emulsion" generally refers to a dispersed system consisting of two or more immiscible liquids, one of which is dispersed in the form of small droplets within the other. These droplets typically have diameters between 100 nanometers and 10 micrometers. The formation of an emulsion generally requires the action of an emulsifier, which reduces interfacial tension, allowing the two originally immiscible liquids to coexist stably. Common emulsions include water-in-oil (W / O) and oil-in-water (O / W).

[0102] As used herein, the term "aqueous agent" generally refers to a uniform liquid mixture formed by dissolving or dispersing other substances in water as the primary solvent. The solute in the aqueous agent can be various inorganic compounds, organic compounds, etc. Water, as a solvent, has good solubility and dispersibility, enabling the solute to be evenly distributed therein. The properties of the aqueous agent depend on the type and concentration of the solute and the properties of the solvent (water).

[0103] As used herein, the term "chelating agent" is a class of organic compounds that can form complexes by coordinating with metal ions. It has a stronger binding ability with metal ions than water molecules and can form relatively stable complexes. The structure of the chelating agent usually contains multiple functional groups that can form complexes with metals, such as carboxyl groups, alcoholic hydroxyl groups, thiol groups, amine groups, etc. Commonly used chelating agents include disodium EDTA, diethylenetriamine pentaacetic acid (DTPA), n,n,n',n'-tetrakis (carboxymethyl) ethylenediamine, NTA, tetraacetic acid ethylenediamine (EGTA), N,N,N',N'-tetrakis (2-pyridinecarboxylic acid) ethylenediamine (TPEN), etc.

[0104] As used herein, the term "thickener," also known as a gelling agent, is a substance that increases the viscosity of latex or liquids. Common thickeners include carbomer, xanthan gum, polyacrylate crosspolymer-6, sodium polyacrylate, polyvinyl pyrrolidone, methylcellulose, and hydroxyethylcellulose.

[0105] As used herein, the term "emulsifier" is a substance that can form a stable emulsion from a mixture of two or more immiscible components. Common cosmetic emulsifiers include PEG-20 methyl glucoside sesquistearate, methyl glucoside sesquistearate, steareth-2, steareth-21, ceteareth-2, ceteareth-21, beheneth-25, PEG-7 sodium olive oil carboxylate, ceteareth-6 olive oil esters, cetearyl alcohol, PEG-7 hydrogenated castor oil, PEG-40 castor oil, polyglyceryl-2 dipolyhydroxystearate, and the like.

[0106] As used herein, the term "emollient" refers to a cosmetic ingredient that helps keep the skin soft, flexible, and smooth. Common emollients include polydimethylsiloxane (PDMS), triethylhexanoin, and the like.

[0107] As used herein, the term "humectant" generally refers to a class of substances that can absorb water from the surrounding environment or prevent water loss, thereby increasing or maintaining the moisture content in the system. Humectants often have hydrophilic functional groups, such as hydroxyl, carboxyl, amide, etc. These functional groups can form hydrogen bonds or other types of bonds with water molecules, thereby effectively capturing and retaining water. Common humectants include polyols (such as glycerol, propylene glycol), natural moisturizing factors (such as amino acids, urea, etc.), hyaluronic acid, etc., which are widely used in cosmetics, medicine, food and other fields.

[0108] As used herein, the term "penetration enhancer" generally refers to a substance that can increase the penetration of drugs or other active ingredients through biological barriers such as the skin and mucous membranes. Penetration enhancers can change the physical structure of biological barriers, such as increasing their porosity, reducing their lipid order, etc., thereby promoting the penetration of substances. Common penetration enhancers include surfactants, alcohols, fatty acids, etc., which have important applications in the fields of medicine and cosmetics. The penetration enhancers used in the present invention include phytol, tetrahydropiperine and tridecapeptide-1, etc. As used herein, the term "skin moisturizer" is a class of mild lipophilic substances that can make the skin softer and tougher. In addition to lubricating the skin, it can also cover the skin, reduce water evaporation from the skin surface, diffuse moisture from the basal tissue to the stratum corneum, induce further hydration of the stratum corneum, preserve the moisture of the skin itself, and complete the emollient effect. The scope of moisturizers is very wide, including various oils, fats and waxes, alkanes, fatty acids, fatty alcohols and their esters, natural animal and plant oils, fatty acid glycerides, etc.

[0109] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific embodiments. Example

[0110] Example 1. Construction of an integrated plasmid expression system for elastin peptide and its fermentation production

[0111] Construction of integration plasmid

[0112] The elastin peptide (SEQ ID NO: 1) is a fragment (amino acids 85-184) of human elastin (UniProt ID P15502) obtained by screening elastin. To facilitate purification, a His tag (-GSSHHHHHH, SEQ ID NO: 30) was attached to the C-terminus. The peptide was designed for secretory expression by the yeast Komagataella phaffii. A signal peptide from the yeast mating factor α (SEQ ID NO: 2) of Saccharomyces cerevisiae was placed upstream of the elastin peptide sequence to direct secretion. To improve expression efficiency, the product gene sequence was designed and optimized using codon optimization technology.

[0113] The specific method is as follows: The nucleotide sequence encoding the signal peptide and the elastin peptide with a C-terminal His tag was codon-optimized using the "Codon Optimization Tool" available on the website of Integrated DNA Technologies, Inc. The codon-optimized gene sequence (SEQ ID NO: 3) was then synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0114] An integrative plasmid was used to integrate the elastin peptide expression cassette into the yeast genome. The elastin peptide expression cassette included the methanol-inducible promoter of the AOX1 gene (SEQ ID NO: 4, Komagataella phaffii CBS 7435, chromosome IV, nucleotides 238,036-238,974, GenBank LT962479.2), the synthetic gene sequence encoding the elastin peptide as described above (SEQ ID NO: 3), and the transcriptional terminator of the AOX1 gene (SEQ ID NO: 5, Komagataella phaffii CBS 7435, chromosome IV, nucleotides 240,967-241,316, GenBank LT962479.2).

[0115] The integration plasmid backbone includes: a geneticin (G418) resistance gene cassette (SEQ ID NO: 6), a Col E1 replicon (SEQ ID NO: 7), and an ampicillin resistance gene cassette (SEQ ID NO: 8). The backbone sequence was synthesized by Beijing Liuhe BGI Genomics Co., Ltd. (SEQ ID NO: 9).

[0116] To construct the integrating plasmid, the backbone sequence (SEQ ID NO: 9) was amplified using primers backbone-F and backbone-R (SEQ ID NO: 10 and SEQ ID NO: 11).

[0117] Genomic DNA of the BG11 strain (PS10011) from BioGrammatics Inc. was prepared using the TIANamp Yeast DNA Extraction Kit (DP307-02) from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0118] Using this genomic DNA as a template, the methanol-inducible promoter sequence (SEQ ID NO: 4) of the AOX1 gene was amplified using primers Pro-F and Pro-R (SEQ ID NO: 12 and SEQ ID NO: 13); the transcription terminator (SEQ ID NO: 5) of the AOX1 gene was amplified using primers Ter-F and Ter-R (SEQ ID NO: 16 and SEQ ID NO: 17).

[0119] The codon-optimized elastin peptide gene sequence synthesized by Beijing Liuhe BGI was used as a template to amplify the elastin peptide gene (SEQ ID NO: 3) using primers ELN-F and ELN-R (SEQ ID NO: 14 and SEQ ID NO: 15).

[0120] The amplified products were ligated using NEB's NEBuilder HiFi DNA Assembly Premix Kit. The ligated products were transformed into Escherichia coli DH5α (DL1003M, Shanghai Weidi Biotechnology Co., Ltd.). Transformed E. coli clones were screened on Luria-Bertani (LB) medium containing 100 mg / L ampicillin sodium. The assembled plasmid sequence was verified by Sanger sequencing. This plasmid was designated pELN1 (SEQ ID NO: 18, Figure 1).

[0121] Construction of elastin peptide expression strain

[0122] The integrated plasmid pELN1 was purified from E. coli culture using a plasmid miniprep kit (DP103-02) from Tiangen Biochemical Technology (Beijing) Co., Ltd. The plasmid was linearized using the PmeI restriction enzyme (ThermoFisher ER1341).

[0123] 100 ng of linearized plasmid was used to transform the BG11 strain (PS10011, BioGrammatics Inc.) by electroporation. Preparation of competent cells and electroporation followed the protocol in Expression of proteins in Pichia pastoris (Methods in Enzymology, 2021 Vol 660, 53-80).

[0124] Transformants were screened on YPD solid medium containing 1 g / L G418.

[0125] The integration of the integrated plasmid into the transformant genome was verified by colony PCR using primer 5U (SEQ ID NO: 19), which binds to the genomic sequence upstream of the methanol-inducible promoter of the AOX1 gene, and primer 3C (SEQ ID NO: 20), which binds to the plasmid-specific sequence downstream of the elastin expression vector. The integrated transformant is expected to produce a 2.1kb PCR product (Figure 2). The PCR product was sequenced by Sanger to confirm the presence of the elastin expression vector. The steps of colony PCR are also derived from the article Expression of proteins in Pichia pastoris (Methods in Enzymology, 2021 Vol 660, 53-80). The verified transformant strain was named Strain-ELN.

[0126] Protein fermentation process

[0127] The fermentation medium (see Table 1) was sterilized at 121°C for 30 minutes and cooled to 28°C for later use. PTM1 and HMP were added after cooling. The fermentation medium was prepared as shown in Tables 1 and 2.

[0128] Table 1 Fermentation medium composition

[0129] Table 2 PTM1 formulation

[0130] The strain Strain-ELN single clone was added to more than 50 mL of YPD medium (1% yeast extract powder, 2% peptone, 2% glucose) and cultured overnight at 28°C and 250 rpm (first-stage shake flask seeds). The next day, the first-stage shake flask seeds were transferred to more than 100 mL of YPD medium, and the initial OD 600 =0.5, 28 ° C, 250 rpm culture for 24 hours as the tank seed. The tank seed was added to the fermentation tank containing the above fermentation medium, and the initial OD 600 = 0.3, and the fermentation culture was carried out for 22-24 hours. Glycerol was added and the cells were cultured until the wet weight reached 330-380 g / L. Then, methanol was switched to induce the culture for 170 hours. The fermentation protocol was carried out according to the Pichia Fermentation Process Guidelines provided by Thermofisher (https: / / tools.thermofisher.com / content / sfs / manuals / pichiaferm_prot.pdf).

[0131] Protein purification

[0132] Take the cultured bacteria, centrifuge the bacterial solution in a low-temperature centrifuge, and retain the supernatant.

[0133] The supernatant obtained above was filtered through a 0.45 μm filter membrane and then passed through a 5 ml nickel ion affinity chromatography column for protein purification. The steps are as follows:

[0134] 1) The sample was flowed through the Ni column at a rate of 1 ml / min;

[0135] 2) Equilibrate the column with Buffer A (20 mM PB, pH 7.5);

[0136] 3) Elution with 20 mM, 300 mM, and 500 mM imidazole.

[0137] 4) The samples eluted in each step were subjected to SDS-PAGE gel analysis to determine whether the target protein was present.

[0138] The results are shown in FIG3 , which shows that the target protein was obtained by the method of Example 1.

[0139] Example 2. 0.01% recombinant human elastin formula

[0140] The recombinant human elastin peptide purified in Example 1 is formulated at 0.01% and 99.99%. The formulation comprises the following ingredients by weight: 0.05 parts by weight of a chelating agent, 0.1 parts by weight of a thickener, 3.0 parts by weight of a polyol, 2 parts by weight of a vegetable oil, 1 part by weight of a high-melting-point fatty compound, 3 parts by weight of a skin moisturizer, 2 parts by weight of an emulsifier, 1 part by weight of a preservative, a pH adjustment system to 6.0, and the remainder made up to 100 parts by weight with water.

[0141] Example 3.2% recombinant human elastin peptide formula

[0142] The recombinant human elastin peptide purified in Example 1 is prepared in a 2% to 98% formulation. The formulation comprises the following ingredients by weight: 0.5 parts by weight of a chelating agent, 0.2 parts by weight of a thickener, 4.0 parts by weight of a polyol, 3 parts by weight of a vegetable oil, 2 parts by weight of a high-melting-point fatty compound, 8 parts by weight of a skin moisturizer, 6 parts by weight of an emulsifier, 2 parts by weight of a preservative, a pH adjustment system to 6.5, and the remainder made up to 100 parts by weight with water.

[0143] Example 4.5% recombinant human elastin peptide formula

[0144] The recombinant human elastin peptide purified in Example 1 is prepared in a 5% or 95% formulation. The formulation comprises the following ingredients by weight: 1 part by weight of a chelating agent, 0.5 parts by weight of a thickener, 6.0 parts by weight of a polyol, 5 parts by weight of a vegetable oil, 3 parts by weight of a high-melting-point fatty compound, 20 parts by weight of a skin moisturizer, 10 parts by weight of an emulsifier, 3 parts by weight of a preservative, a pH adjustment system to 7.0, and the remainder made up to 100 parts by weight with water.

[0145] The chelating agent in the above embodiments 2-4 can be EDTA2Na, the thickener can be carbomer or xanthan gum, the polyol can be glycerol or pentylene glycol, the vegetable oil can be shea butter, the high melting point fatty compound can be cetearyl alcohol, cetyl ethylhexanoate, cetearyl alcohol, cetyl ethylhexanoate, the skin moisturizer can be a cetearyl glucoside mixture, the emulsifier can be glyceryl stearate, PEG-100 stearate mixture, triethylhexanoin, and the preservative can be p-hydroxyacetophenone.

[0146] Example 5: Efficacy Verification of Recombinant Human Elastin Peptide

[0147] Gene expression test methods:

[0148] Primary human fibroblasts (HDFs) were purchased from Cell Technology, cultured under conditions of 95% air, 5% CO2, and 37°C. When reaching a density of 80-90%, HDF cells were digested from the culture medium and seeded into 12-well plates at a density of 100,000 cells per well. After 48 hours, the cells were treated with different concentrations (3ppm, 15ppm, 100ppm) of the test sample (recombinant elastin peptide purified in Example 1) (n=3), and the HDF cells were exposed to UVA irradiation of 5J / cm2 for 24 hours. After 24 hours of treatment, the HDF cells were rinsed twice with cooled PBS buffer, and then total RNA was extracted from the cell lysate using Qiagen's RNeasy Mini Kit. The total RNA was reverse transcribed into cDNA for amplification. The expression of the target gene was detected by quantitative PCR (qPCR) (primers are shown in Table 3). The relative gene expression level was calculated by the 2-ΔΔCt method.

[0149] Table 3 Primers for quantitative PCR detection of target genes

[0150] Figure 4 is a graph showing the promoting effect of recombinant elastin peptide on elastin gene expression in fibroblasts, wherein, as the concentration of recombinant elastin (ELN) increases, the gene expression effect of elastin increases significantly, reaching a high value at 15ppm. Figure 5 is a graph showing the promoting effect of recombinant elastin peptide on collagen VII (COL7A1) gene expression, wherein, as the concentration of recombinant elastin (ELN) increases, the gene expression effect of collagen VII increases significantly, showing concentration dependence. Figure 6 is a graph showing the promoting effect of recombinant elastin peptide on collagen IV (COL4A1) gene expression, wherein, as the concentration of recombinant elastin (ELN) increases, the gene expression effect of collagen IV increases significantly, showing concentration dependence. Figure 7 is a UVA irradiation comparison graph, wherein, after UVA irradiation, the gene expression of collagen I in fibroblasts is significantly reduced, wherein, after fibroblasts treated with recombinant elastin (ELN), the gene expression of collagen I is restored; proving that recombinant elastin has significant UVA protection efficacy;

[0151] Clinical efficacy testing methods:

[0152] Thirty-five healthy female volunteers aged 30 to 60 years (whose skin conditions met any two of the following criteria: canthus wrinkles, grades 2 to 4; eye wrinkles, grades 2 to 5; and nasolabial folds, grades 1 to 3; and whose left and right cheeks both had F4 values ​​greater than 6 or R2 values ​​less than 0.65) were randomly assigned to a 2% recombinant elastin emulsion (i.e., the product of Example 3) or a placebo (the placebo differed from Example 3 in that the recombinant elastin was replaced with water) for 56 consecutive days. The placebo and sample (i.e., the 2% recombinant elastin emulsion) groups were randomly assigned to apply to half of their face twice daily. Skin moisture content was measured using a CM825 (CK) instrument on D0, D14, D28, and D56, and skin color, skin gloss, and crow's feet were measured using a VISIA-CR instrument to analyze the effects of the samples on skin condition.

[0153] FIG8 is a comparison chart of skin moisture content after 14 days of use of an emulsion formula containing 60 ppm of recombinant elastin peptide. The skin moisture content of the 2% ELN (i.e., the product of Example 4 of the present invention) was twice as high as that of the control group (placebo). The effect became more significant with increasing usage time.

[0154] Figure 9 shows a comparison of skin glossiness after 14 days of use using an emulsion formula containing 60 ppm of recombinant elastin peptide. The 2% ELN (i.e., the product of Example 3 of the present invention) significantly increases skin glossiness, and with increasing use time, the skin glossiness can be doubled after two months.

[0155] FIG10 is a comparison chart showing crow's feet wrinkles after 14 days of use using an emulsion formula containing 60 ppm of recombinant elastin peptide. 2% ELN (i.e., the product of Example 3 of the present invention) significantly reduces crow's feet wrinkles, and the effect becomes more pronounced with increasing usage time.

[0156] FIG11 is a comparison chart of the skin L value after two months of use of an emulsion formula containing 60 ppm of recombinant elastin peptide. The L value of the skin of the 2% ELN (i.e., the product of Example 3 of the present invention) was significantly increased compared to the control group (placebo), indicating that the formula has a certain whitening effect.

[0157] Example 6 Construction of an expression system for recombinant human elastin peptide

[0158] Further, an expression system for the recombinant human elastin peptide sequenced as SEQ ID NO: 21 was constructed. The construction method involved retaining the C-terminal His tag design of the expression plasmid pELN1 disclosed in Example 1, i.e., a His tag (-GSSHHHHHH) of SEQ ID NO: 30 was added to the C-terminus of SEQ ID NO: 21. The sequence of the recombinant human elastin peptide-His tag fusion protein was SEQ ID NO: 29. Using pELN1 (SEQ ID NO: 18) as a template, the recombinant human elastin peptide coding sequence (SEQ ID NO: 31) was amplified by PCR using primers ELN2-F (SEQ ID NO: 22) and ELN2-R (SEQ ID NO: 23). Using pELN1 as a template, primers BB-F (SEQ ID NO: 24) and BB-R (SEQ ID NO: 25) were used to PCR amplify a plasmid backbone containing the coding sequences of the AOX1 promoter, Saccharomyces cerevisiae α mating factor signal peptide, AOX1 transcription terminator, geneticin (G418) resistance gene cassette, Col E1 replicon, and ampicillin resistance gene cassette.

[0159] The amplified products were ligated using NEB's NEBuilder HiFi DNA Assembly Premix Kit. The ligated products were transformed into Escherichia coli DH5α (DL1003M, Shanghai Weidi Biotechnology Co., Ltd.). Transformed E. coli clones were screened on Luria-Bertani (LB) medium supplemented with 100 mg / L ampicillin sodium. The assembled plasmid sequence was verified by Sanger sequencing. This plasmid was designated pELN2 (Seq ID NO: 26, Figure 12).

[0160] The integrated plasmid pELN2 was purified from E. coli culture using a plasmid miniprep kit (DP103-02) from Tiangen Biochemical Technology (Beijing) Co., Ltd. The plasmid was linearized using the PmeI restriction enzyme (ThermoFisher ER1341).

[0161] 100 ng of the linearized plasmid was used to transform the BG11 strain (PS10011, BioGrammatics Inc.) by electroporation. Competent cells were prepared and electroporated according to the protocol in Expression of proteins in Pichia pastoris (Methods in Enzymology, 2021 Vol 660, 53-80).

[0162] Transformants were screened on YPD solid medium containing 1 g / L G418.

[0163] The integration of the integrated plasmid into the transformant genome was verified by colony PCR using primer 5U2 (SEQ ID NO.27), which binds to the genomic sequence upstream of the methanol-inducible promoter of the AOX1 gene, and primer 3C2 (SEQ ID NO.28), which binds to the elastin peptide coding sequence. The integrated transformant is expected to produce a 1.4kb PCR product. The PCR product was sequenced by Sanger to confirm the presence of the elastin expression vector. The colony PCR steps are also derived from the article Expression of proteins in Pichia pastoris (Methods in Enzymology, 2021 Vol 660, 53-80). The verified transformant strain was named Strain-ELN2.

[0164] FIG13 is an electrophoretic diagram of PCR verification of the insertion of the recombinant human elastin peptide integration plasmid pELN2 into the transformant genome in an embodiment of the present invention.

[0165] The protein fermentation process and protein purification process were the same as in Example 1. The fermentation product was detected by SDS-Page, and the results are shown in FIG14 .

[0166] Example 7 Clinical efficacy test

[0167] Healthy female volunteers aged 30 to 60 years (whose skin conditions met any two of the following criteria: canthus wrinkles, grades 2-4; eye wrinkles, grades 2-5; and nasolabial folds, grades 1-3; and whose left and right cheeks had an average F4 value greater than 6 or an R2 value ≤ 0.65) were selected. They were randomly assigned to a lotion formula containing 60 ppm recombinant elastin and a placebo formula for 56 consecutive days. The placebo and sample groups were randomly assigned to a half-face application twice daily. Dermal density, skin elasticity, and skin firmness were measured using the VISIA-CR instrument after 14, 28, and 56 days of use.

[0168] The emulsion formulation used the recombinant human elastin peptide (SEQ ID NO: 29) purified in Example 6 and prepared with reference to the formulation in Example 3. The placebo formulation was prepared by replacing the recombinant human elastin in the emulsion with water.

[0169] Figure 18 shows a comparison of dermal density after 14, 28, and 56 days of use of an emulsion containing 60 ppm of recombinant elastin peptide (compared to a control group). Using the 60 ppm emulsion significantly increased skin density after just 14 days, and the effect increased with increasing use. After 28 and 56 days of use, the increase in skin density was significantly greater than that of the control group.

[0170] Figure 19 shows the skin elasticity of the emulsion containing 60 ppm of recombinant elastin peptide after 14, 28, and 56 days of use (compared to the control group). The skin elasticity of the emulsion containing 60 ppm of recombinant elastin peptide gradually increased over time, and the numerical value showed that it was better than the control group.

[0171] Figure 20 shows the skin firmness of the emulsion containing 60 ppm of recombinant elastin peptide after 14, 28, and 56 days of use (compared to the control group). Skin firmness increased with the use of the emulsion containing 60 ppm of recombinant elastin peptide, far exceeding that of the control group.

[0172] Figure 21 is a comparison chart of the skin roughness of the emulsion formula containing 60ppm recombinant elastin peptide of the present invention after 14 days, 28 days, and 56 days of use (compared with the control group). The improvement of skin roughness increased with time when using the emulsion containing 60ppm recombinant elastin peptide, and the data after 14 days were significantly better than those of the control group. Example 8 Skin Permeability Study of Recombinant Human Elastin Peptide

[0173] The recombinant human elastin peptide contains an 89-amino acid sequence (SEQ ID NO: 29) and a molecular weight of approximately 8 kd. To verify the transdermal properties of the recombinant human elastin peptide, the inventors labeled the recombinant human elastin peptide with FITC and conducted transdermal experiments using an artificially constructed 3D skin model (EpiKutis).

[0174] 8.1 Fluorescent Labeling of Recombinant Human Elastin Peptide

[0175] Dissolve the recombinant human elastin peptide in sodium carbonate buffer, slowly add FITC solution dropwise, and incubate the reaction solution at 4°C in the dark for 8 h after the required FITC is added. Add NH4Cl solution (1 M) to the reaction solution, shake gently to evenly mix, and terminate the reaction at 4°C for 2 h.

[0176] The markers were separated chromatographically using a Sephadex G25 filtration column. After equilibration with 5 to 10 column volumes of PBS (10 mM, pH 7.4), 1 mL of the reaction mixture was injected from the top of the column. The column was opened and allowed to flow completely into the column bed. The sample was then rinsed three times with a small amount of PBS buffer (10 mM, pH 7.4) and eluted with PBS buffer (10 mM, pH 7.4). Two color bands appeared during the elution: FITC-recombinant human elastin peptide and FITC, from bottom to top.

[0177] A column volume of sample was collected to draw an elution curve, and the eluate was collected at 0.5 mL / tube, with a total of 220 tubes collected. The absorbance and fluorescence values ​​at 495 nm (excitation wavelength 495 nm, emission wavelength 525 nm) were tested for each tube, and the elution curve was drawn with the elution volume as the horizontal axis and the absorbance / fluorescence value as the vertical axis (Figure 15). The results showed that the elution curves drawn with absorbance and fluorescence values ​​basically overlapped and showed two elution peaks. The first peak at 10-15 mL was FITC-elastin, and the second peak washed out after 40 mL was free FITC. The two elution peaks were completely separated, indicating that FITC-elastin was successfully separated and purified. After collecting 10-15 mL of eluate, it was freeze-dried to obtain a solid FITC-recombinant human elastin peptide conjugate.

[0178] 8.2 Preparation of test samples

[0179] Recombinant human elastin peptide emulsion and aqueous solution were prepared according to the formulas in Table 4 and Table 5, respectively.

[0180] Table 4 Elastin peptide emulsion

[0181] Table 5 Recombinant elastin aqueous solution

[0182] 8.3Resuscitation and cultivation of 3D skin models

[0183] The established 3D skin (EpiKutis) was placed in a 6-well plate containing 0.9 mL of culture medium and cultured in a CO2 incubator for 1 h. The 3D skin was transferred to a 6-well plate containing 0.9 mL of EpiGrowth culture medium (Guangdong Boxi Biotechnology Co., Ltd.) and cultured in a CO2 incubator for 18 h before conducting the study.

[0184] 8.4 Transdermal Experimental Steps and Results

[0185] A 0.2 mg / mL FITC-recombinant human elastin peptide aqueous solution (aqueous solution), a 0.2 mg / mL FITC-elastin emulsion (see Table 4), and a 0.2 mg / mL FITC-recombinant human elastin peptide aqueous solution (see Table 5) were prepared. After adding 25 μL of each sample to the 3D skin surface in a clean bench, 0.9 mL of culture medium (EpiGrowth culture medium) was used as the receiving medium and placed in a CO2 incubator for 3D skin observation. At 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12 hours, 200 μL of the receiving solution was collected for fluorescence detection, and the same volume of isothermal blank culture medium (EpiGrowth culture medium) was added at the same time. The fluorescence value of each sample was measured using a microplate reader (excitation wavelength 495 nm, emission wavelength 530 nm). The fluorescence quantitative standard curve is shown in Figure 16.

[0186] Table 6 Sample 3D skin model transdermal data

[0187] Table 6 shows the test results for each sample on the 3D skin model. The elastin aqueous solution and recombinant human elastin peptide aqueous solution demonstrated superior permeation performance compared to the recombinant human elastin peptide emulsion. Both solutions showed a FITC-elastin fluorescence signal in the receiving cell 8 hours after administration, with a permeation rate of approximately 0.2% after 12 hours. For the recombinant human elastin peptide emulsion, the FITC-elastin fluorescence signal remained below the limit of quantification for the first 10 hours after administration, and the cumulative permeation after 12 hours was 0.12%.

[0188] 8.5 Confocal Laser Scanning Microscope (CLSM) Observation

[0189] Following the aforementioned 3D skin model penetration investigation method, three samples of recombinant human elastin peptide aqueous solution, recombinant human elastin peptide emulsion, and recombinant human elastin peptide aqueous solution were treated with the 3D skin for 12 hours. The 3D skin was then frozen and sectioned, stained with DAPI, and the distribution of FITC-elastin within the skin was examined using CLSM (Figures 17A-C). The fluorescence signal of each sample group was primarily concentrated in the stratum corneum. After 12 hours of treatment with each sample, a small amount of fluorescence signal was observed in the epidermis of the 3D skin. The fluorescence signal of the elastin aqueous solution was the strongest (see Figure 17C), indicating that this formulation of recombinant human elastin peptide has the best penetration effect.

[0190] At the same time, those skilled in the art should understand that the present invention is not limited to the above-described embodiments. The above-described embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant human elastin peptide with anti-aging efficacy, characterized by: The recombinant human elastin peptide is selected from the following a) to c): a) comprising the amino acid sequence shown in SEQ ID NO: 1; b) comprising a fragment of the amino acid sequence shown in SEQ ID NO: 1, wherein the fragment comprises 30% or more of SEQ ID NO: 1; c) mutants of the above a) and b).

2. The recombinant human elastin peptide according to claim 1, comprising the amino acid sequence of SEQ ID NO: 21 or a variant sequence obtained by inserting, substituting or deleting one or more amino acids in SEQ ID NO: 21; the variant sequence has at least 90% identity with SEQ ID NO: 21; preferably, the variant retains anti-aging efficacy equivalent to that of SEQ ID NO: 21; preferably, the anti-aging efficacy is selected from one or more of the following: 1) promoting the expression of endogenous elastin; 2) promoting the expression of collagen IV and / or VII genes in the dermal-epidermal junction; 3) protecting collagen I from damage by ultraviolet rays; 4) increasing or maintaining skin moisture content; 5) improving skin gloss; 6) reducing crow's feet; 7) increasing the L value of the skin; 8) increasing skin density; 9) improving skin elasticity or firmness.

3. The recombinant human elastin peptide with anti-aging efficacy according to claim 1 or 2, wherein the N-terminus and / or C-terminus of the recombinant human elastin peptide sequence comprises a fusion protein sequence.

4. The recombinant human elastin peptide with anti-aging efficacy according to claim 3, wherein the recombinant human elastin peptide sequence comprises a protein tag at the N-terminus and / or the C-terminus.

5. The recombinant human elastin peptide with anti-aging efficacy according to claim 4, wherein the protein tag is one of a His tag, a glutathione S-transferase tag, a maltose binding protein tag, a SUMO tag, a NusA tag, a TrxA tag, and a DsbA tag; preferably, the recombinant human elastin peptide comprises the amino acid sequence shown in SEQ ID NO:

29. 6 . The recombinant human elastin peptide with anti-aging efficacy according to claim 1 , wherein the recombinant human elastin peptide is expressed by a host. 7 . The recombinant human elastin peptide with anti-aging efficacy according to claim 6 , wherein the recombinant human elastin peptide is expressed by a bacterial host.

8. The recombinant human elastin peptide with anti-aging efficacy according to claim 7, wherein the bacterial host is one of Escherichia coli, Bacillus subtilis and Rhodococcus erythropolis.

9. The recombinant human elastin peptide with anti-aging efficacy according to claim 6, wherein the recombinant human elastin peptide is expressed by a fungal host. 10 . The recombinant human elastin peptide with anti-aging efficacy according to claim 9 , wherein the fungal host is one of Pichia pastoris, Saccharomyces cerevisiae and Aspergillus oryzae.

11. The recombinant human elastin peptide with anti-aging efficacy according to claim 6, wherein the recombinant human elastin peptide is expressed by a eukaryotic cell host. 12 . The recombinant human elastin peptide with anti-aging efficacy according to claim 11 , wherein the eukaryotic cell is one of insect cells, CHO cells, mouse cells and human cells. 13 . The recombinant human elastin peptide with anti-aging efficacy according to claim 6 , wherein the recombinant human elastin peptide is expressed in host cells. 14 . The recombinant human elastin peptide with anti-aging efficacy according to claim 6 , wherein the recombinant human elastin peptide is exocrinely expressed by host cells. 15 . The recombinant human elastin peptide with anti-aging efficacy according to claim 14 , wherein the recombinant human elastin peptide is exocrine expressed, and its precursor protein comprises a secretion signal peptide sequence.

16. The recombinant human elastin peptide with anti-aging efficacy according to claim 14, wherein the recombinant human elastin peptide is exogenously expressed by either Pichia pastoris or Saccharomyces cerevisiae.

17. The recombinant human elastin peptide with anti-aging efficacy according to claim 15, wherein the precursor protein comprises a signal peptide sequence of Saccharomyces cerevisiae mating factor α, and the amino acid sequence of the signal peptide of mating factor α is shown in SEQ ID NO:

2.

18. The recombinant human elastin peptide with anti-aging efficacy according to claim 1 or 2, wherein the gene encoding the recombinant human elastin peptide comprises a fragment encoding the elastin peptide in the cDNA sequence of the human elastin gene.

19. The recombinant human elastin peptide with anti-aging efficacy according to claim 1 or 2, wherein the gene encoding the recombinant human elastin peptide is an artificially synthesized gene.

20. The recombinant human elastin peptide with anti-aging efficacy according to claim 15, wherein the recombinant human elastin peptide is exocrine-expressed, and the gene encoding the precursor of the recombinant human elastin peptide comprises a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding the recombinant human elastin peptide. 21 . The recombinant human elastin peptide with anti-aging efficacy according to claim 20 , wherein the gene sequence encoding the recombinant human elastin peptide is a codon-optimized artificial synthetic gene sequence.

22. The recombinant human elastin peptide with anti-aging efficacy according to claim 15, wherein the recombinant human elastin peptide is exocrine-expressed, and the gene encoding the precursor of the recombinant human elastin peptide comprises a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding the recombinant human elastin peptide, and a nucleotide sequence encoding a C-terminal His tag.

23. The recombinant human elastin peptide with anti-aging efficacy according to claim 22, wherein the artificially synthesized gene encoding the elastin peptide is synthesized by codon-optimizing the nucleotide sequence encoding the signal peptide, the elastin peptide, and the C-terminal His tag. 24 . The recombinant human elastin peptide with anti-aging efficacy according to claim 23 , wherein the artificial synthetic gene encoding the elastin peptide comprises the nucleotide sequence shown in SEQ ID NO: 3 or 31.

25. An isolated nucleic acid, characterized in that Encoding the recombinant human elastin peptide according to any one of claims 1 to 24.

26. A carrier, characterized in that Comprising the isolated nucleic acid of claim 25.

27. A host cell, characterized in that Comprising the nucleic acid of claim 25 or the vector of claim 26.

28. The method for preparing the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 24, characterized in that: The gene encoding the recombinant human elastin peptide or its precursor protein is expressed through a promoter; the promoter is an inducible promoter or a constitutive promoter.

29. A method for preparing the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 24 from a fermentation culture, characterized in that: The following steps are involved: 1) Fermenting a host cell containing a gene encoding a recombinant human elastin peptide or a precursor protein thereof to obtain a fermentation product; preferably, the host cell is as described in claim 27. 2) Protein purification to obtain recombinant human elastin peptide.

30. The method for preparing a recombinant human elastin peptide with anti-aging efficacy from a fermentation culture according to claim 29, wherein for a fermentation product expressing intracellularly, the recombinant human elastin peptide is prepared from a cell lysate; for a fermentation product expressing exocrinely, the recombinant human elastin peptide is prepared from a fermentation supernatant; Preferably, the cell lysate or fermentation supernatant is filtered through a 0.45 μm filter membrane, and then the protein is purified through an affinity chromatography column to obtain the recombinant human elastin peptide with anti-aging efficacy.

31. A recombinant human elastin peptide composition with anti-aging efficacy, characterized by: The composition comprises 0.01% to 5% of the recombinant human elastin peptide according to any one of claims 1 to 24, and the rest is a formulating agent.

32. A recombinant human elastin peptide composition with anti-aging efficacy, characterized in that: The composition comprises a recombinant human elastin peptide and excipients; preferably, the recombinant human elastin peptide comprises the amino acid sequence shown in SEQ ID NO: 21; preferably, the composition is an emulsion, an aqueous solution or an aqueous solution.

33. The composition according to claim 32, which is an aqueous solution, wherein the excipients include a solvent, a humectant, a penetration enhancer and a solubilizing agent; preferably, the solvent is water; preferably, the humectant includes propylene glycol; preferably, the penetration enhancer includes tetrahydropiperine and / or tridecapeptide-1; preferably, the solubilizing agent includes PEG-40 hydrogenated castor oil.

34. The composition according to claim 32, which is an emulsion, wherein the excipient comprises one or more selected from a solvent, a chelating agent, a thickening agent, an emulsifier, an emollient, a humectant, a penetration enhancer, and a pH regulator; preferably, the solvent is water; preferably, the chelating agent is disodium EDTA; preferably, the thickening agent comprises one or more of carbomer, xanthan gum, and polyacrylate crosspolymer-6; preferably, the emulsifier is selected from methyl glucose sesquistearate and / or PEG-20 methyl glucoside sesquistearate; preferably, the emollient is selected from polydimethylsiloxane and / or triethylhexanoin; preferably, the penetration enhancer is selected from one or more of phytol, tetrahydropiperine, and tridecapeptide-1; preferably, the humectant comprises propylene glycol.

35. Use of the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 24, or the composition according to any one of claims 31 to 34 in the preparation of skin care products, skin repair dressings, implants, artificial skin, medical devices and / or biomaterials.

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