Use of recombinant collagen in the preparation of a preparation for increasing the expression of skin anti-wrinkle related genes

Abstract

The present application relates to the use of recombinant collagen in the preparation of a preparation for increasing the expression of skin anti-wrinkle related genes, wherein the amino acid sequence of the recombinant collagen comprises the sequence shown in SEQ ID NO. 1. The present application detects the expression of a series of skin anti-wrinkle related genes by transcriptomics, and proves that the recombinant collagen provided by the present application can promote the expression of skin cell anti-wrinkle genes, and can be used for the development and application of various cosmetic products, and endows the cosmetic products with the efficacy and effect of promoting skin anti-wrinkle ability. The above-mentioned genes can also be used as a reference for evaluating the skin anti-wrinkle ability of other raw materials or cosmetics in the future.

Description

Technical Field

[0001] This invention relates to the field of cosmetic formulations, and more particularly to the application of recombinant collagen in the preparation of formulations that increase the expression of skin anti-wrinkle related genes. Background Technology

[0002] Collagen is an important structural protein whose main function is to provide structural support for the body, maintain tissue stability and elasticity, and play a vital role in maintaining normal physiological functions and overall health. With the development of genetic engineering technology, people have begun to try to produce collagen through recombinant DNA technology. Through genetic engineering, target genes can be inserted into expression vectors, and the target protein can be expressed in the expression host.

[0003] Recombinant collagen is a type of collagen produced by using the amino acid sequence of in vivo collagen as a template. This sequence is designed, enzymatically digested, and spliced, then transferred into engineered cells, primarily *E. coli* and *Pichia pastoris*, using synthetic biology techniques. The rapid production capacity of these engineered cells is then utilized. If human collagen is used as a template, the resulting recombinant collagen is called recombinant human collagen. For example, CN109988243A discloses a recombinant human type III collagen α1 chain and its applications, which, from the amino terminus, sequentially includes: an amino-terminal affinity purification tag, a mature human type III collagen peptide chain, and a carboxyl-terminal affinity purification tag.

[0004] Compared to animal-derived collagen extracted using traditional methods, recombinant collagen exhibits better water solubility and processability. It also boasts advantages such as single-component composition, controllable preparation process, and short production cycle, making product quality easier to control. As a biocompatible and biodegradable polymer, recombinant collagen holds broad application prospects in the medical field, offering numerous benefits from promoting wound healing and tissue regeneration to serving as a drug carrier and in cosmetic skincare.

[0005] In summary, providing a novel recombinant collagen and applying it to skincare and beauty applications such as anti-wrinkle treatment has become one of the most pressing issues to be addressed in this field. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of recombinant collagen in the preparation of formulations that increase the expression of skin anti-wrinkle related genes. Through transcriptomics, the expression of a series of skin anti-wrinkle related genes was detected, demonstrating that the recombinant collagen provided by this invention can promote the expression of skin cell anti-wrinkle genes.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the use of recombinant collagen in the preparation of formulations that increase the expression of skin anti-wrinkle related genes, wherein the amino acid sequence of the recombinant collagen includes the sequence shown in SEQ ID NO.1.

[0009] SEQ ID NO.1:GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGT AGEPGRDGNPGSDGLPGRDSPGGKGDRGENGSPGAPGAPGHPGPPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPP.

[0010] The recombinant collagen provided by this invention can promote the expression of skin anti-wrinkle related genes and can be used in the development and application of various cosmetic products, giving cosmetic products the efficacy and function of promoting skin anti-wrinkle ability.

[0011] Preferably, the skin anti-wrinkle related genes include any one or a combination of at least two of the following: collagen-encoding genes, FGF family genes, TIMP family genes, GDF family genes, ELN genes, TGFB1 genes, or RARA genes.

[0012] In bioinformatics analysis, a log2 fold change value of 0.25 is typically used as the threshold for screening differentially expressed genes, which translates to a relative expression level of approximately 1.19. A relative expression level greater than 1.19 is considered a promotion. The recombinant collagen provided by this invention can promote the expression of skin anti-wrinkle-related genes. After application, the overall log2 fold change of these skin anti-wrinkle-related genes increases by approximately 0.948, meaning gene expression is promoted by approximately 1.929 times, demonstrating its effect in promoting the expression of skin anti-wrinkle-related genes.

[0013] Collagen in the skin is a key component for maintaining skin structure, elasticity, and strength. The recombinant collagen provided by this invention promotes the expression of collagen-encoding genes, thus promoting collagen expression. FGF is the gene encoding fibroblast growth factor, which promotes cell growth, differentiation, and repair, contributing to skin regeneration and repair, and maintaining skin firmness and elasticity. TIMP is the gene encoding extracellular matrix protease inhibitor, which inhibits the activity of matrix metalloproteinases (MMPs), preventing excessive degradation of collagen and elastin, thereby delaying wrinkle formation. GDF is a gene encoding... The genes encoding growth differentiation factor (GGF) belong to the TGF-β superfamily and participate in regulating cell differentiation and growth, playing a role in the repair and maintenance of skin tissue. The ELN gene is the gene encoding elastin, which gives skin elasticity and extensibility; the loss of elastin is an important factor in wrinkles caused by aging. The TGFB1 gene is the gene encoding transforming growth factor β1, a multifunctional cytokine that regulates collagen synthesis and extracellular matrix remodeling. The RARA gene is the gene encoding retinoic acid (a derivative of vitamin A), which promotes skin cell renewal and repair by binding to its receptor, reducing fine lines and wrinkles.

[0014] Preferably, the collagen encoding gene includes any one or a combination of at least two of COL4A1, COL6A3, COL6A6, COL7A1, COL8A1, COL11A2, COL13A1, COL16A1, COL17A1, COL23A1, or COL27A1.

[0015] Preferably, the FGF family genes include any one or a combination of at least two of FGF1, FGF4, FGF5, FGF7, FGF17, FGF19, FGF21 or FGF22.

[0016] Preferably, the TIMP family genes include TIMP3 and / or TIMP4.

[0017] Preferably, the GDF family genes include any one or a combination of at least two of GDF5, GDF9, or GDF15.

[0018] Preferably, the formulation includes pharmaceuticals and / or cosmetics.

[0019] Preferably, the cosmetic includes any one or a combination of at least two of the following: facial cleanser, serum, eye cream, toner, lotion, face cream, or face mask.

[0020] Preferably, the concentration of recombinant collagen in the formulation is 0.1 to 1 mg / mL (e.g., it can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, etc.).

[0021] Secondly, the present invention provides an expression promoter that increases the expression level of skin anti-wrinkle related genes, wherein the expression promoter is recombinant collagen, and the amino acid sequence of the recombinant collagen includes the sequence shown in SEQ ID NO.1.

[0022] Thirdly, the present invention provides the application of products for detecting the expression level of biomarkers in evaluating the skin's anti-wrinkle ability in cosmetics or their raw materials, wherein the biomarkers are skin anti-wrinkle related genes, and the skin anti-wrinkle related genes include any one or a combination of at least two of the following: COL4A1, COL6A3, COL6A6, COL7A1, COL8A1, COL11A2, COL13A1, COL16A1, COL17A1, COL23A1, COL27A1, FGF1, FGF4, FGF5, FGF7, FGF17, FGF19, FGF21, FGF22, TIMP3, TIMP4, GDF5, GDF9, GDF15, ELN, TGFB1, or RARA.

[0023] This invention provides a combination of skin anti-wrinkle related genes that can be used as a biomarker to evaluate the anti-wrinkle ability of other raw materials or cosmetics in the future.

[0024] Preferably, the product for detecting the expression level of biomarkers includes any one or a combination of at least two of primer combinations, reagents, or detection models.

[0025] Preferably, the nucleic acid sequence of the primer combination includes the sequences shown in SEQ ID NO.5 to SEQ ID NO.58.

[0026] The primers for a series of genes provided by this invention can be quickly used in qPCR experiments to verify the anti-wrinkle ability of new raw materials or products on the skin, while ensuring the accuracy of the experimental results.

[0027] The nucleic acid sequence information of the primer combinations provided by this invention is shown in Table 1.

[0028] Table 1

[0029]

[0030]

[0031]

[0032] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) This invention provides a new recombinant collagen that has a better ability to promote the expression of skin anti-wrinkle related genes than existing collagen, and can be added to skin care products as a raw material to achieve better skin care effects.

[0035] (2) This invention provides a set of genes that promote anti-wrinkle effects on the skin and provides primers that can be used to detect the above genes, which can be used as a reference for evaluating the anti-wrinkle ability of other raw materials or cosmetics in the future. Attached Figure Description

[0036] Figure 1 A heatmap showing gene expression in the experimental and control groups. Detailed Implementation

[0037] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0038] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0039] Example 1

[0040] This embodiment provides a recombinant collagen that increases the expression of skin anti-wrinkle related genes. The amino acid sequence of the recombinant collagen is shown in SEQ ID NO.1. The preparation method of the recombinant collagen includes the following steps:

[0041] 1. Fermentation process

[0042] First, the bacterial strain was activated by streaking it onto YPD agar plates and incubating it at 30°C for 72 hours. Next, seed culture preparation was performed by selecting healthy single colonies in a clean bench and inoculating them into Erlenmeyer flasks containing YPD agar, then incubating them on a shaker at 30°C and 250 rpm for 30 hours. Finally, the seed culture was transferred to a fermenter containing 40 g / L glycerol and 4.35 mL / L BSM agar for fermentation.

[0043] Fermentation process control includes the following steps:

[0044] (1) Initial glycerol consumption stage: The temperature is maintained at 30℃, the pH is maintained at 5.5 with ammonia, the air flow rate is 2vvm, the dissolved oxygen is stirred and maintained at 20%-40%, and after the dissolved oxygen value soars, the additional glycerol supplementation stage begins.

[0045] (2) Glycerin Supplementation Stage: Maintain temperature at 30℃, pH at 5.5 using ammonia, and air flow rate at 2 vvm; add 80-100 mL of 50% glycerin. Observe the rate of change in DO to control the flow rate, and simultaneously monitor OD. 600 OD pending 600 When the growth reaches 250, stop adding glycerol and starve the culture for about 1 hour.

[0046] (3) Methanol induction stage: control the temperature at 25℃ and pH at 6, add 30-50 mL of pure methanol at a flow rate of 35 mL / h; observe the change of DO to judge the flow rate, and finally stabilize the flow rate at 10 mL of methanol per liter of culture medium per hour, based on the premise that methanol does not accumulate. Fermentation ends after 96-120 h of pure methanol induction.

[0047] 2. Purification process

[0048] (1) The obtained fermentation broth was first filtered through a 0.45 μm ceramic membrane and then purified by hydrophobic chromatography (Dongfulong hydrophobic chromatography column Phevl Purose 6FF; PrePack50 / 200). The sample and column specifications are shown in Table 2.

[0049] Table 2

[0050]

[0051]

[0052] (2) Preparation of experimental materials and preparation of buffer solution:

[0053] Distilled water: 5L;

[0054] Equilibration buffer: 20mM PB, 1M ammonium sulfate, pH 7, 5L;

[0055] Elution buffer: 20 mM PB, pH 7, 5 L;

[0056] Washing buffer: 1M NaOH, 1L;

[0057] The above buffer solution must be filtered through a 0.45 μm filter membrane.

[0058] (3) The purification experiment includes the following steps:

[0059] 1) Wash the chromatography column 3CV (column volume, hereinafter omitted) with equilibration buffer at a linear flow rate of 90 cm / h until the baselines for pH, UV, and conductivity detection are stable and consistent with the equilibration buffer.

[0060] 2) Load the sample and equilibration buffer at a rate of 1:1 at 60 cm / h (20 mL / min), collect the flow-through components during the loading process, and detect the purity and content of the target protein.

[0061] 3) After loading the sample, wash the 2CV with equilibration buffer at a speed of 90 cm / h (30 mL / min) to remove unbound material until the UV detection value drops to the initial value.

[0062] 4) Elute the target protein by mixing the elution buffer and equilibration buffer at a ratio of 6:4 at a speed of 90 cm / h (30 mL / min), and collect the elution fraction.

[0063] 5) Wash the chromatographic column with at least 2 column volumes of 1M NaOH at a speed of 60 cm / h (20 mL / min) to remove residual contaminants in the packed column, such as lipids, endotoxins, and nucleic acids.

[0064] 6) Wash the chromatography column with 5-10 CV of distilled water at a rate of 60 cm / h (20 mL / min) until the effluent is neutral in pH and has a conductivity close to 0, in order to remove 1 M NaOH.

[0065] 7) Wash and store with 20% ethanol in 3CV to prevent microbial growth.

[0066] Example 2

[0067] This embodiment uses transcriptomics sequencing to study the effect of recombinant collagen on the expression levels of skin anti-wrinkle related genes in Example 1. Recombinant collagen was added to the cell culture medium of adherent human skin fibroblasts (BJ cells) at a final concentration of 1 mg / mL. After 24 hours of culture, the cells were lysed using Trizon sequencing, and the lysate was sent to BGI Genomics for transcriptome sequencing. The analysis protocol is shown below.

[0068] 1. Analysis Plan

[0069] 1.1 Species name: Homo sapiens; Source: NCBI; Reference genome version: GCF_000001405.40_GRCh38.p14.

[0070] 1.2 Differential Grouping Settings

[0071] One of the key analytical points in transcriptome sequencing is the comparison of differences in expression levels. The experimental group consisted of BJ cells treated with recombinant collagen, while the control group consisted of BJ cells not treated with recombinant collagen.

[0072] 2. Experimental Procedure

[0073] 2.1 mRNA library construction workflow

[0074] 1) Total RNA is processed using mRNA enrichment or rRNA removal methods; mRNA enrichment: mRNA with polyA tails is enriched using magnetic beads with OligodT; rRNA removal: rRNA is hybridized with a DNA probe, the DNA / RNA hybrid chain is selectively digested with RNase H, the DNA probe is digested with DNase I, and the desired RNA is obtained after purification.

[0075] 2) The obtained RNA was fragmented using a fragmentation buffer, and reverse transcribed using random N6 primers to synthesize cDNA double strands to form double-stranded DNA.

[0076] 3) The ends of the synthesized double-stranded DNA are padded and phosphorylated at the 5' end. A sticky end with an "A" protrusion is formed at the 3' end, and then a bubble-shaped adapter with a protruding "T" at the 3' end is attached.

[0077] 4) The ligation product is amplified by PCR using specific primers.

[0078] 5) The PCR product is denatured into single strands by heat, and then a bridging primer is used to circularize the single-stranded DNA to obtain a single-stranded circular DNA library.

[0079] 6) Sequencing on the machine.

[0080] 4. Data Analysis and Results

[0081] 4.1 Data Analysis Process

[0082] The raw data obtained from sequencing is called raw reads. First, low-quality reads, those contaminated with adapters, and those with excessively high levels of unknown base N are filtered out. The filtered data is called clean reads. These clean reads are then aligned to a reference genome, followed by new transcriptome prediction, SNP & InDel detection, and differentially spliced ​​gene detection. After obtaining new transcripts, those with protein-coding potential are added to the reference gene sequence to form a complete reference sequence, and gene expression levels are calculated. Finally, for multiple samples, differentially expressed genes are detected among different samples according to requirements, and in-depth cluster analysis and functional enrichment analysis are performed on the differentially expressed genes, etc.

[0083] 4.2 The obtained transcriptomics sequencing results retained data with p-values ​​> 0.05, and the FPKM values ​​and log2 fold change values ​​of skin anti-wrinkle related genes were extracted in the experimental and control groups.

[0084] 4.3 The FPKM values ​​of the above genes in the experimental and control groups were integrated into the same table, and the results are shown in Table 3. Heatmap analysis using the Gedeo Bioinformatics Cloud tool yielded the following results: Figure 1 The heatmap shown.

[0085] Table 3

[0086]

[0087]

[0088] As shown in Table 3, the final calculated overall increase in log2 fold change was approximately 0.948, which means that gene expression was promoted by about 1.929 times, proving that recombinant collagen can increase the expression of skin anti-wrinkle related genes.

[0089] Example 3

[0090] This embodiment uses the recombinant collagen provided in Example 1 to verify the effect of different concentrations of recombinant collagen on the expression levels of skin anti-wrinkle related genes, including the following steps:

[0091] 1. Cell sample culture

[0092] Collagen was prepared into test solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, respectively, and added in equal volumes to the cell culture medium of BJ cells that were adhering to the wall and growing, and cultured for 24 h.

[0093] 2. Extraction and purification of total RNA from cells

[0094] (1) Take the cultured cells, remove the culture medium, wash twice with pre-cooled PBS, add 1 mL TRIzol reagent, pipette and collect into a centrifuge tube, and let stand at room temperature for 5 min.

[0095] (2) Add 200 μL of chloroform to the centrifuge tube, shake the centrifuge tube vigorously for 15 s, let it stand for 10 min, and then centrifuge at 12000g for 15 min at 4℃.

[0096] (3) After centrifugation, the liquid separates into layers. The RNA is present in the upper colorless and transparent aqueous phase layer with a volume of about 600 μL. After transferring to a new centrifuge tube, add 500 μL of isopropanol, slowly invert the tube several times to mix, let stand for 15 minutes, and then centrifuge at 12000 g for 10 min at 4℃.

[0097] (4) After centrifugation, the RNA precipitates at the bottom of the centrifuge tube and appears as a gel-like precipitate when facing the light source. After removing the supernatant, add 1 mL of 75% ethanol to resuspend and wash the RNA, and centrifuge at 7500 g for 5 min at 4°C.

[0098] (5) Remove the ethanol supernatant, let stand for 5 minutes to dry the RNA, and add 20 μL of DEPC-water to dissolve the RNA.

[0099] 3. RNA concentration determination

[0100] (1) Take 1 μL of the extracted RNA and add it to 99 μL of DEPC-water to make a total volume of 100 μL. Add the diluted RNA solution to a 96-well UV plate.

[0101] (2) Place the 96-well plate into a full-wavelength microplate reader, select the area to be tested, and set the program as follows: shake for 10 s, measure the absorbance at 260 nm and 280 nm, and read the values.

[0102] (3) Calculate the OD260 / OD280 ratio. A ratio between 1.8 and 2.0 indicates that the quality of the extracted RNA meets the requirements for subsequent processing.

[0103] (4) The concentration of the RNA solution was calculated according to the formula OD260×0.04×dilution factor, in μg / μL. The RNA concentration was adjusted to 1 μg / μL by diluting with DEPC-water.

[0104] 4. Reverse transcription

[0105] (1) Using a reverse transcription kit (Novizan One-Step Reverse Transcription Kit R333), a reverse transcription system was prepared in a 200 μL PCR tube, containing: a) 1 μg / μL RNA solution, volume 1 μL; b) 5×qRT SuperMix, volume 4 μL; c) premixed enzyme solution, volume 1 μL; d) DEPC-water, volume 14 μL. The final volume of the reverse transcription system was 20 μL. The mixture was gently mixed by pipetting and then briefly centrifuged for 30 s using a handheld centrifuge.

[0106] (2) Place the reverse transcription PCR tube into the RCR instrument and set the reaction program as follows: a) incubate at 50℃ for 15 min; b) inactivate briefly at 85℃ for 10 s, and the mRNA in the system is reverse transcribed into cDNA.

[0107] 5. Quantitative Real-Time PCR

[0108] (1) Using the reagents from the SYBR Green Real-Time PCR Kit, prepare the reaction system in an eight-tube PCR apparatus, including: a) 10 μL of 2×SYBR Green PCR premix; b) 1.5 μL each of forward and reverse primers; c) 1 μg / μL cDNA solution; and d) 6 μL of sterile Grade I water. The final volume of the PCR system is 20 μL. Centrifuge briefly for 30 s using a handheld centrifuge.

[0109] (2) Each sample requires three replicate wells for each molecule to be tested, and a blank control without cDNA should be set up; in addition to the molecule to be tested, actin β (ACTB) is also required as a reference molecule.

[0110] (3) Place the eight-tube PCR instrument into a real-time PCR instrument and set the reaction program as follows: a) 95℃ for 2 min; b) 95℃ for 5 min; c) 60℃ for 10 s; d) 20℃ for storage. Repeat steps b and c for 45 cycles.

[0111] (4) After the PCR program is completed, the software automatically generates the CT value for each sample.

[0112] 6. Quantitative analysis of results

[0113] (1) Take the average of the three replicate CT values ​​for each sample and calculate the difference between the CT value of the molecule to be tested and the CT value of the reference molecule (ACTB), i.e., ΔCT.

[0114] (2) Calculate the difference between the ΔCT value of the molecule to be tested in the cell after raw material treatment and the ΔCT value of the molecule to be tested in the untreated cell, i.e., ΔΔCT.

[0115] (3) Calculate the exponent with base 2 and exponent (-ΔΔCT), i.e., 2 -△△CT 2 parallel groups of each concentration -△△CT The average value was taken as the quantitative result of the molecule to be tested in each sample, so as to achieve quantitative detection at the gene level. The test results of skin anti-wrinkle related genes are shown in Table 4.

[0116] Table 4

[0117]

[0118] As shown in Table 4, after treating BJ cells with recombinant collagen at concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, the average fold increase in the relative expression levels of 27 genes, including COL4A1, was greater than 1.19. The addition of recombinant collagen promoted the expression of skin wrinkle-related genes. Specifically, the 0.5 mg / mL concentration ensured that the expression of all wrinkle-related genes was promoted and that the relative expression levels were the highest.

[0119] Example 4

[0120] In this embodiment, the recombinant collagen provided in Example 1, as well as collagen 1, collagen 2, and collagen 3, were used to determine the expression levels of skin anti-wrinkle related genes, referring to Example 3. The results are shown in Table 5. The amino acid sequence of collagen 1 is shown in SEQ ID NO.2, the amino acid sequence of collagen 2 is shown in SEQ ID NO.3, and the amino acid sequence of collagen 3 is shown in SEQ ID NO.4.

[0121] SEQ ID NO.2: LPGYKGEPGRDGDKGDRGLPGFPGLHGMGSKGEMGAKGDKGSPGF YGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGASPGEPGYMGLP GIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVRGAIGS KGESGVDGLMGPAGPKGQPGDPGPQGPPGLDGKPGRE.

[0122] SEQ ID NO.3: GPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNY SPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAP。

[0123] SEQ ID NO.4: GFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGM RGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNT。

[0124] Table 5

[0125]

[0126]

[0127] As can be seen from Table 5, compared with other types of collagen, the recombinant collagen provided by this invention has the strongest promoting effect on the expression of skin anti-wrinkle related genes, and has the application prospect of preparing cosmetics with anti-wrinkle function.

[0128] Application Example 1

[0129] This application example uses the recombinant collagen provided in Example 1 to prepare cosmetics with anti-wrinkle functions, such as facial cleansers, serums, eye creams, toners, lotions, creams, or masks.

[0130] Application 1, preparation of a face cream, comprising the following components by weight percentage: 0.1% recombinant collagen, 7% caprylic / capric triglyceride, 5% liquid paraffin, 4% glycerin, 3% propylene glycol, 3% ethylhexyl palmitate, 3% petrolatum, 2% cetearyl alcohol ether-21, 2% dimethicone, 2% cetearyl alcohol, 1.5% cetearyl alcohol ether-2, 1.5% glyceryl monostearate, 0.2% methylparaben, 0.2% carbomer, 0.2% triethanolamine, 0.1% ethylparaben, with the balance being water (65.2%).

[0131] Application 2, preparation of the emulsion, comprising the following components by mass percentage: 0.1% recombinant collagen, 5% glycerol, 5% hydrogenated polyisobutylene, 4% ethylhexyl palmitate, 3% propylene glycol, 3% caprylic / capric triglyceride, 2% polydimethylsiloxane, 1.5% cetearyl ether-21, 1.2% cetearyl ether-2, 0.5% tocopherol, 0.2% methylparaben, 0.1% triethanolamine, 0.1% ethylparaben, 0.1% carbomer, with the balance being water (74.2%).

[0132] Application 3, preparation of toner, comprising the following components by mass percentage: 0.1% recombinant collagen, 5% glycerin, 3% propylene glycol, 0.2% diazolidinyl urea, and the balance being water (91.7%).

[0133] In summary, this invention, through transcriptomics, detected the expression of a series of genes related to skin anti-wrinkle properties, demonstrating that the recombinant collagen provided by this invention can promote the expression of anti-wrinkle genes in skin cells. This recombinant collagen can be used in the development and application of various cosmetic products with anti-wrinkle effects, endowing these products with the efficacy and function of promoting skin anti-wrinkle capabilities. Furthermore, the combination of skin anti-wrinkle-related genes provided by this invention can also serve as a reference for future evaluation of the anti-wrinkle capabilities of other raw materials or cosmetics.

[0134] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. The application of recombinant collagen in the preparation of cosmetics that increase the expression levels of skin anti-wrinkle related genes, characterized in that, The amino acid sequence of the recombinant collagen is the sequence shown in SEQ ID NO.1; The skin anti-wrinkle related genes are any one or a combination of at least two of the following: collagen encoding genes, FGF family genes, TIMP family genes, GDF family genes, ELN genes, TGFB1 genes, or RARA genes. The collagen encoding gene is any one or a combination of at least two of COL4A1, COL6A3, COL6A6, COL7A1, COL8A1, COL11A2, COL13A1, COL16A1, COL17A1, COL23A1 or COL27A1; The FGF family genes are any one or a combination of at least two of FGF1, FGF4, FGF5, FGF7, FGF17, FGF19, FGF21 or FGF22; The TIMP family genes are TIMP3 and / or TIMP4; The GDF family gene is any one or a combination of at least two of GDF5, GDF9, or GDF15.

2. The application according to claim 1, characterized in that, The cosmetics include any one or a combination of at least two of the following: facial cleanser, serum, eye cream, toner, lotion, face cream, or face mask.

3. The application according to claim 1, characterized in that, The concentration of recombinant collagen in the cosmetic product is 0.1~1 mg / mL.

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