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

Abstract

The present invention relates to the field of biotechnology and discloses a recombinant human elastin peptide with anti-aging efficacy and its application in a composition thereof. The recombinant human elastin peptide comprises the elastin peptide amino acid sequence SEQ ID: 1 or a fragment thereof, wherein the fragment comprises 30% of SEQ ID: 1, or a fragment comprises more than 30% of SEQ ID: 1, or a mutant of the above sequence. The recombinant human elastin peptide can be directed to synthesize a protein of a highly effective fragment, with a 100% human homologous sequence, while being low-cost, easy to mass-produce, avoiding animal sources, and avoiding viral risks. This allows the recombinant human elastin peptide to be safely used in cosmetics as an anti-aging and anti-photoaging ingredient, while also providing support for its application in the field of biomedical materials.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant human elastin peptide with anti-aging efficacy and application of a composition thereof. Background Art

[0002] 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 key role in skin aging. Its degradation and destruction can lead to symptoms such as decreased skin elasticity, sagging, and wrinkles.

[0003] 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. As described in CN202211035433.1, a method for synthesizing elastin using Escherichia coli (VPTGIG) 25 A repeat sequence method is described, which is simple to purify and provides a new type of drug carrier. CN202010611388.4 discloses a fragment of the elastin repeat sequence (VAPGVG) 3S synthesized by Escherichia coli, which is repeated 3-7 times in tandem and has been shown to have high superoxide radical scavenging activity.

[0004] 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. Summary of the Invention

[0005] 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.

[0006] The present invention discloses a recombinant human elastin peptide with anti-aging efficacy. The recombinant human elastin peptide comprises the elastin peptide amino acid sequence SEQ ID: 1 or a fragment thereof, wherein the fragment comprises 30% of SEQ ID: 1, or the fragment comprises more than 30% of SEQ ID: 1, or a mutant of the above sequence.

[0007] In a preferred technical solution, the N-terminus and / or C-terminus of the recombinant human elastin peptide sequence comprises a fusion protein sequence.

[0008] In a preferred technical solution, the fusion protein sequence is a protein tag.

[0009] 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.

[0010] 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.

[0011] 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 cell, CHO cell, mouse cell and human cell.

[0012] In a preferred technical solution, the recombinant human elastin peptide is expressed in host cells.

[0013] In a preferred technical solution, the recombinant human elastin peptide is exogenously expressed by host cells.

[0014] 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.

[0015] In a preferred technical solution, the recombinant human elastin peptide is exogenously expressed by either Pichia pastoris or Saccharomyces cerevisiae.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In a preferred technical solution, the nucleotide sequence of the artificial synthetic gene encoding elastin peptide is shown in Seq ID NO.3.

[0023] 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.

[0024] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is expressed via an inducible promoter.

[0025] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is expressed by a constitutive promoter.

[0026] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is introduced into the host cell.

[0027] 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.

[0028] In a preferred technical solution, the gene encoding the recombinant human elastin peptide or its precursor protein is integrated into the host genome.

[0029] 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:

[0030] 1) fermenting a host cell containing a gene encoding a recombinant human elastin peptide or a precursor protein thereof;

[0031] 2) Protein purification: obtain recombinant human elastin peptide from the fermentation product.

[0032] 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.

[0033] 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.).

[0034] 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;

[0035] 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.

[0036] As a preferred technical solution, the affinity chromatography column protein purification steps are as follows:

[0037] S1, flow the sample through the Ni column at a rate of 1 ml / min;

[0038] S2. Equilibrate the column with Buffer A.

[0039] S3, elution with 20 mM, 300 mM, and 500 mM imidazole;

[0040] S4. The eluted samples were subjected to SDS-PAGE gel analysis to determine whether the target protein was present.

[0041] 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.

[0042] The formulation includes the following components in percentage by weight: 0.05 to 1 parts by weight of a chelating agent, 0.1 to 0.5 parts by weight of a thickener, 3.0 to 6.0 parts by weight of a polyol, 2 to 5 parts by weight of vegetable oils and fats, 1 to 3 parts by weight of a high-melting-point fatty compound, 3 to 20 parts by weight of a skin moisturizer, 2 to 10 parts by weight of an emulsifier, 1 to 3 parts by weight of a preservative, a pH adjustment system to 6.0 to 7.0, and the remainder is made up to 100 parts by weight with water.

[0043] A recombinant human elastin peptide composition with anti-aging efficacy is used in the preparation of skin care products, skin repair dressings, implants, artificial skin, medical devices, and biomaterials.

[0044] Advantages of the present invention:

[0045] The present invention utilizes Pichia yeast fermentation to synthesize a functional fragment of human elastin, which can significantly promote the synthesis of its own elastin gene and fibronectin gene, and can also significantly promote the synthesis of collagen IV and VII genes in the dermal-epidermal junction layer.

[0046] UV protection experiments have shown that this recombinant human elastin fragment can protect elastin, collagen I, IV, and VII from UV damage. Clinical trials have confirmed that a lotion containing 1% of this recombinant elastin fragment can significantly improve dermal density and elasticity, while also significantly improving skin hydration and roughness.

[0047] The present invention adopts gene recombination technology, accesses the genome of Pichia pastoris, utilizes the expression system of Pichia pastoris, and efficiently synthesizes a continuous 100 amino acid fragment of elastin starting from the 85th amino acid, and then obtains high-purity recombinant elastin by separation and purification.

[0048] This method enables the targeted synthesis of highly effective fragments with 100% human homology, while also being cost-effective, easy to mass-produce, and avoiding animal sources and viral hazards. This allows the safe use of 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

[0049] Figure 1This is a comprehensive plasmid construction diagram of the recombinant elastin of the present invention;

[0050] Figure 2 This is a diagram showing cPCR verification of the expression of elastin fragments in the integrated plasmid according to an embodiment of the present invention;

[0051] Figure 3 This is an SDS-Page display of the bands of the recombinant elastin after fermentation and purification according to an embodiment of the present invention;

[0052] Figure 4 This figure shows the promoting effect of the 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.

[0053] Figure 5 This figure shows the promoting effect of recombinant elastin on collagen VII gene expression in the present invention. As the concentration of recombinant elastin (ELN) increases, the gene expression effect of collagen VII increases significantly, showing concentration dependence.

[0054] Figure 6 This figure shows the promoting effect of recombinant elastin 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.

[0055] Figure 7 This 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. However, after fibroblasts treated with recombinant elastin (ELN), the gene expression of collagen I is restored, demonstrating that recombinant elastin has significant UVA protection efficacy.

[0056] Figure 8 This is a comparison chart of skin moisture content after 14 days of use of an emulsion formula containing 60 ppm recombinant elastin in the present invention's experiment. The skin moisture content of the 2% ELN (i.e., the product of Example 3 of the present invention) was twice as high as that of the control group (placebo). The effect became more significant with increasing use time.

[0057] Figure 9 This is a comparison chart of skin glossiness after 14 days of use of the emulsion formula containing 60 ppm recombinant elastin in the present invention. Among them, the skin glossiness of 2% ELN (i.e., the product of Example 3 of the present invention) increased significantly. Moreover, with the increase in usage time, the skin glossiness can be doubled after two months.

[0058] Figure 10This is a comparison chart of crow's feet wrinkles after 14 days of use using an emulsion formula containing 60 ppm recombinant elastin in the present invention. Among them, 2% ELN (i.e., the product of Example 3 of the present invention) has a very significant effect in reducing crow's feet wrinkles. The effect becomes more obvious with increasing use time.

[0059] Figure 11 This is a comparison chart of the skin L value after two months of use of the emulsion formula containing 60 ppm recombinant elastin in the present invention. The L value of the skin using 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. DETAILED DESCRIPTION

[0060] 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.

[0061] 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.

[0062] Example 1:

[0063] Construction of an integrated plasmid expression system for recombinant elastin

[0064] The elastin peptide (Seq ID NO. 1) is a fragment of human elastin (UniProt ID P15502) (amino acids 85-184). To facilitate purification, a His tag (-GSSHHHHHH) was attached to the C-terminus. This peptide was designed for secretory expression by the yeast Komagataella phaffii. A signal peptide (Seq ID NO. 2), amino acids 1-89, from the yeast 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 techniques.

[0065] 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" on the website of Integrated DNA Technologies, Inc. The codon-optimized gene sequence (Seq ID No. 3) was then synthesized by BGI TECH SOLUTIONS (BEIJING LIUHE) CO., LIMITED.

[0066] A comprehensive 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 previously (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).

[0067] 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 BGI TECH SOLUTIONS (BEIJING LIUHE) CO., LIMITED (Seq ID NO. 9).

[0068] To construct the integration 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).

[0069] The genomic DNA of the BG11 strain (PS10011) of BioGrammatics Inc. was prepared using the TIANamp Yeast DNA Extraction Kit (DP307-02) of TIANGEN BIOTECH (BEIJING) Co., LTD.

[0070] 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).

[0071] The synthetic elastin peptide gene (Seq ID NO. 3) was amplified using primers ELN-F and ELN-R (Seq ID NO. and Seq ID NO. 15).

[0072] The transcription terminator of the AOX1 gene (Seq ID NO. 5) was amplified using genomic DNA of the BG11 strain (PS10011) from BioGrammatics Inc. as a template using primers Ter-F and Ter-R (Seq ID NO. 16 and Seq ID NO. 17).

[0073] The amplified products were ligated using NEB's NEBuilder HiFi DNA Assembly Premix Kit. Transformed E. coli DH5α (DL1003M from WEIDI Bio, Inc.) clones were screened on Luria-Bertani (LB) medium containing 100 mg / L ampicillin sodium. The assembled sequence of the plasmid was verified by Sanger sequencing. The plasmid was named pELN1 (Seq ID NO. 18, Figure 1 )

[0074] Construction of recombinant elastin expression strain

[0075] The integration plasmid pELN1 was purified from E. coli culture using the TIANprep Mini Plasmid Kit (DP103-02) from Tianjin Biotechnology (Beijing) Co., Ltd. The plasmid was linearized using the PmeI restriction enzyme (ThermoFisher ER1341), which cuts in the middle of the methanol-inducible promoter sequence of the AOX1 gene.

[0076] 100 ng of the linearized plasmid was used to transform the BG11 strain (PS10011, BioGrammatics Inc.) by electroporation. Preparation of competent cells and electroporation followed the protocol in "Protein Expression in Pichia pastoris" (Methods in Enzymology, 2021 Vol 660, 53-80).

[0077] Transformants were screened on YPD solid medium containing 1 g / L G418.

[0078] Integration of the integrated plasmid into the AOX1 gene methanol-inducible promoter site of the transformant genome was verified by colony PCR using primer 5U (Seq ID NO. 19), which binds to the genomic sequence upstream of the AOX1 gene methanol-inducible promoter, and primer 3C (Seq ID NO. 20), which binds to the plasmid-specific sequence downstream of the elastin expression vector ( Figure 2 ). The expected integrated transformant should produce a 2.1 kb PCR product ( Figure 2The presence of the elastin expression vector was confirmed by Sanger sequencing of the PCR product. The colony PCR protocol was also adapted from the book "Protein Expression in Pichia pastoris," Methods in Enzymology, 2021, Vol. 660, pp. 53–80. The verified transformant strain was named Strain-ELN.

[0079] Protein fermentation process

[0080] The fermentation medium, including glycerol, calcium sulfate dihydrate, magnesium sulfate heptahydrate, potassium sulfate, ammonium sulfate, defoamer, PTM1, and HMP (hexa-sodium metaphosphate solution), was sterilized at 121° C. for 30 minutes, cooled to 28° C. for later use, and PTM1 and HMP were added after cooling.

[0081] The fermentation medium configuration is shown in Table 1.

[0082] Table 1

[0083]

[0084] The PTM1 configuration is shown in Table 2.

[0085] Table 2

[0086] Reagents Concentration (g / L) brand Quality grade / item number Copper sulfate pentahydrate 6 Sinopharm Shanghai trial AR Potassium iodide (keep away from light) 0.088 Sinopharm Shanghai trial AR Manganese sulfate monohydrate 3 Sigma M7634-500G Sodium molybdate dihydrate 0.2 Sinopharm Shanghai trial AR Boric acid 0.02 Sinopharm Shanghai trial AR Cobalt chloride 0.5 Sinopharm Shanghai trial AR zinc chloride 20 Sinopharm Shanghai trial AR Ferrous sulfate 65 Mackline F903428-1kg Biotin 0.2 Solebao D8150 concentrated sulfuric acid 5ml / /

[0087] A single clone was added to at least 50 mL of culture medium and cultured overnight at 28°C, 250 rpm. The next day, the seed from the primary shake flask was transferred to at least 100 mL of culture medium, cultured at an OD of 0.5, and incubated at 28°C, 250 rpm for 24 hours as the tank seed. The tank seed was added to the aforementioned fermentation medium to an initial OD of 0.3. Fermentation was incubated for 22-24 hours, followed by the addition of glycerol until the cell wet weight reached 330-380 g / L. Induction was then switched to methanol for 170 hours.

[0088] Protein purification process

[0089] Take the cultured bacteria, centrifuge the bacterial solution in a low-temperature centrifuge, and retain the supernatant.

[0090] The supernatant obtained above was filtered through a 0.45 μm filter membrane and then passed through a 5 ml affinity chromatography column for protein purification. The steps are as follows:

[0091] 1) The sample was flowed through the Ni column at a rate of 1 ml / min;

[0092] 2) Equilibrate the column with Buffer A (20 mM PB, pH 7.5);

[0093] 3) Elution with 20 mM, 300 mM, and 500 mM imidazole.

[0094] 4) The eluted samples were subjected to SDS-PAGE gel analysis to determine whether the target protein was present.

[0095] Example 2:

[0096] The 0.01% and 99.99% formulation of recombinant human elastin obtained in Example 1 comprises the following ingredients in percentage 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.

[0097] Example 3

[0098] The 2% and 98% formulations of the recombinant human elastin obtained in Example 1 comprise 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.

[0099] Example 4

[0100] The 5% and 95% formulations of the recombinant human elastin obtained in Example 1 comprise the following ingredients by weight: 1 part by weight of a chelating agent, 0.5 part 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.

[0101] experiment

[0102] Efficacy verification process of recombinant elastin

[0103] Gene expression test methods:

[0104] Primary human fibroblasts (HDFs) were cultured in an atmosphere of 95% air, 5% CO2, and 37°C. When they reached a density of 80-90%, the 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 of the test sample (n=3), and the irradiated HDF cells were exposed to 5J / cm2 of UVA. 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 RNeasyMini Kit. The total RNA was reverse transcribed into cDNA for amplification. The expression of the target gene was measured by quantitative PCR. The relative gene expression level was calculated by the 2-ΔΔCt method.

[0105] Clinical efficacy testing methods:

[0106] 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.

[0107] After the above experiments, the present invention used Pichia pastoris to biosynthesize a 100-amino acid elastin fragment. The present invention verified in vitro that this fragment promotes fibroblast synthesis of elastin, fibronectin, and several collagens, such as collagen types IV and VI, and has potential anti-aging effects. The main characteristics of skin aging are epidermal thinning, flattening of the dermal-epidermal junction, and overall degradation of the extracellular matrix (ECM). Elastic fibers play a vital role in skin aging. Their degradation and destruction can cause loss of skin elasticity, sagging, wrinkle formation, and even the appearance of skin plaques. Elastin, the main component of elastic fibers, reaches very high levels in neonates, then begins to decline, with almost no new elastin production in adulthood. Elastin-derived peptides (EDPs) produced by elastin degradation can participate in regulating various cellular activities, such as cell adhesion, chemotaxis, and proliferation, particularly fragments containing GXXPG.

[0108] Collagen is a core component of the extracellular matrix (ECM), providing support and signaling for cells. The collagen family comprises 28 types in vertebrates and can be divided into several subfamilies based on their supramolecular assembly. These include fibrillar collagens (types I, II, III, V, XI, XXIV, and XXVII), fibril-associated collagens with interrupted triple helices (FACITs, types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII), network collagens (types IV, IV, VI, VIII, and X), beaded filament-forming collagens (types VI, XXVI, and XXVIII), anchoring fibrils (type VII), and transmembrane collagens (types XIII, XVII, XXIII, and XXV). The complexity of the collagen network is further enhanced by the use of different collagen α chains in trimerization and network assembly, post-translational modifications of collagen α chains, and interactions with other ECM proteins. Collagen IV is an important component in maintaining the structure of the basement membrane and provides a scaffold for other basement membrane proteins. Collagen IV interacts with soluble glycoproteins, proteoglycans, and growth factors in a tissue-specific manner, contributing to the functional integrity of the basement membrane.

[0109] The present invention verifies for the first time through in vitro cell experiments that the recombinant elastin can significantly promote the expression of elastin genes, collagen IV and collagen VII genes in fibroblasts, increase the firmness and elasticity of the skin, and strengthen the basement membrane of the skin.

[0110] Clinical trials have shown that a lotion formula containing 60ppm of recombinant elastin can significantly increase skin moisture content, brighten skin tone, improve skin glossiness, and significantly reduce the area of ​​crow's feet compared to a placebo formula.

[0111] Furthermore, ultraviolet irradiation experiments revealed that the recombinant elastin protein can also protect against damage caused by ultraviolet rays. Under ultraviolet irradiation conditions, the expression of the elastin gene in fibroblasts decreased significantly. However, in the group treated with recombinant elastin, elastin gene expression was unaffected and remained at a high level. A similar trend was observed for collagen VII: after ultraviolet irradiation, the collagen VII gene was barely expressed, while the addition of recombinant elastin significantly increased collagen VII expression. This suggests that the recombinant elastin protein has the effect of reducing damage to the skin caused by ultraviolet radiation.

[0112] 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 shown in SEQ ID NO:

1. 2 . The recombinant human elastin peptide according to claim 1 , comprising a protein tag at its N-terminus and / or C-terminus. 3 . The recombinant human elastin peptide according to claim 2 , wherein the protein tag is selected from the group consisting 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. 4 . The recombinant human elastin peptide according to claim 1 , wherein the recombinant human elastin peptide is expressed in a host cell. 5 . The recombinant human elastin peptide according to claim 1 , wherein the recombinant human elastin peptide is expressed by a bacterial host. 6 . The recombinant human elastin peptide according to claim 5 , wherein the bacterial host is one of Escherichia coli, Bacillus subtilis and Rhodococcus erythropolis. 7 . The recombinant human elastin peptide according to claim 1 , wherein the recombinant human elastin peptide is expressed by a fungal host. 8 . The recombinant human elastin peptide according to claim 7 , wherein the fungal host is one of Pichia pastoris, Saccharomyces cerevisiae and Aspergillus oryzae. 9 . The recombinant human elastin peptide according to claim 1 , wherein the recombinant human elastin peptide is exogenously expressed by Pichia pastoris or Saccharomyces cerevisiae.

10. The recombinant human elastin peptide according to any one of claims 1 to 3, wherein the recombinant human elastin peptide is expressed by a eukaryotic cell host. The recombinant human elastin peptide according to claim 10 , wherein the eukaryotic cell is one of an insect cell, a CHO cell, a mouse cell, and a human cell.

12. A method for preparing the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 11 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; 2) Protein purification to obtain recombinant human elastin peptide.

13. Use of a composition comprising the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 11 in the preparation of skin care products or artificial skin.

14. Use of a composition comprising the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 11 in the preparation of a skin repair dressing.

15. Use of a composition comprising the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 11 in the preparation of an implant.

16. Use of a composition comprising the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 11 in the preparation of a medical device.

17. Use of a composition comprising the recombinant human elastin peptide with anti-aging efficacy according to any one of claims 1 to 11 in the preparation of biomaterials.

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