Application of recombinant collagen in preparation of preparation for increasing expression quantity of skin anti-wrinkle related genes

Abstract

The invention relates to an application of recombinant collagen in preparation of a preparation for increasing the expression quantity of a skin anti-wrinkle related gene. The amino acid sequence of the recombinant collagen comprises a sequence as shown in SEQ ID NO.1. The invention also relates to a preparation method of the recombinant collagen. The expression condition of a series of genes related to skin wrinkle resistance is detected in a transcriptomics mode, and it is proved that the recombinant collagen can promote expression of skin cell wrinkle resistance genes, can be used for development and application of various cosmetic products, endows the cosmetic products with the effect and effect of promoting the skin wrinkle resistance, and has a good application prospect. The gene can also be used as a reference for evaluating the anti-wrinkle capability of other raw materials or cosmetics on the skin in the future.

Description

Technical Field

[0001] The present invention relates to the field of cosmetic preparations, and in particular to the application of recombinant collagen in the preparation of preparations for increasing the expression of genes related to skin anti-wrinkle. Background Art

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

[0003] Recombinant collagen is a collagen protein prepared by using the amino acid sequence of collagen in the organism as a template, rationally designing, enzymatically cleaving and splicing it, and then transferring it into engineered cells such as Escherichia coli and Pichia pastoris with the help of synthetic biology technology, and utilizing the rapid production capacity of engineered cells. If human collagen is used as a template, the recombinant collagen designed and spliced ​​is called recombinant human collagen. For example, CN109988243A discloses a recombinant human type III collagen α1 chain and its application, which includes, from the amino end, an amino-terminal affinity purification tag, a mature peptide chain of human type III collagen, and a carboxyl-terminal affinity purification tag.

[0004] Compared with animal-derived collagen extracted by traditional methods, recombinant collagen has better water solubility and stronger processability. It also has the characteristics of single component, controllable preparation process, short production cycle, etc., and the product quality is easier to control. As a biocompatible and biodegradable polymer material, recombinant collagen has broad application prospects in the medical field. From promoting wound healing to tissue regeneration, to drug carriers and beauty and skin care, recombinant collagen has many benefits.

[0005] In summary, providing a new type of recombinant collagen and applying it to beauty and skin care fields such as skin anti-wrinkle has become one of the urgent problems to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides the use of recombinant collagen in the preparation of a preparation for increasing the expression of skin anti-wrinkle related genes. Through transcriptomics, the expression of a series of genes related to skin anti-wrinkle was detected, proving that the recombinant collagen provided by the present invention can promote the expression of anti-wrinkle genes in skin cells.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides the use of recombinant collagen in the preparation of a preparation for increasing the expression of genes related to skin anti-wrinkle, 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 the present invention can promote the expression of genes related to skin anti-wrinkle, and can be used for the development and application of various cosmetic products, giving the cosmetic products the efficacy and effect of promoting the anti-wrinkle ability of the skin.

[0011] Preferably, the skin anti-wrinkle related genes include any one or a combination of at least two of 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 usually used as a threshold for screening differentially expressed genes, which is converted into a relative expression multiple of about 1.19, and a relative expression greater than 1.19 is considered to be promoted. The recombinant collagen provided by the present invention can promote the expression of skin anti-wrinkle related genes. After application, the overall log2 fold change of the skin anti-wrinkle related genes is increased by about 0.948, that is, the gene expression is promoted by about 1.929 times, which has the effect of promoting the expression of skin anti-wrinkle related genes.

[0013] Collagen in the skin is a key component for maintaining the structure, elasticity and strength of the skin. The recombinant collagen provided by the present invention has the function of promoting the expression of collagen coding genes, and has a pro-proliferative effect on the expression of collagen; FGF is a gene encoding fibroblast growth factor, which can promote the growth, differentiation and repair of cells, help the regeneration and repair of the skin, and maintain the firmness and elasticity of the skin; TIMP is a gene encoding an extracellular matrix protease inhibitor, which can inhibit the activity of matrix metalloproteinases (MMPs), prevent the excessive degradation of collagen and elastin, and thus delay the formation of wrinkles; GDF is a gene encoding a fibroblast growth factor, which can promote the growth, differentiation and repair of cells, help the regeneration and repair of the skin, and maintain the firmness and elasticity of the skin; TIMP is a gene encoding an extracellular matrix protease inhibitor, which can inhibit the activity of matrix metalloproteinases (MMPs), prevent the excessive degradation of collagen and elastin, and thus delay the formation of wrinkles; The ELN gene is the coding gene for growth differentiation factor, which belongs to the TGF-β superfamily and is involved in regulating cell differentiation and growth, and plays a role in the repair and maintenance of skin tissue; the ELN gene is the coding gene for elastin, which gives the skin elasticity and stretchability. The lack of elastin is an important factor in wrinkled skin in aging; the TGFB1 gene is the coding gene for transforming growth factor β1, which is a multifunctional cytokine that regulates the synthesis of collagen and the remodeling of the extracellular matrix; the RARA gene is the coding gene for retinoic acid (a derivative of vitamin A), which promotes the renewal and repair of skin cells and reduces fine lines and wrinkles by binding to its receptors.

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

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

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

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

[0018] Preferably, the preparation comprises a pharmaceutical and / or a cosmetic.

[0019] Preferably, the cosmetics include any one of facial cleanser, essence, eye cream, lotion, emulsion, facial cream or facial mask, or a combination of at least two of them.

[0020] Preferably, the concentration of recombinant collagen in the preparation is 0.1-1 mg / mL (for example, 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] In a second aspect, the present invention provides an expression promoter for increasing the expression 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] In a third aspect, the present invention provides an application of a product for detecting the expression level of a biomarker in evaluating the ability of cosmetics or their raw materials to increase the anti-wrinkle ability of the skin, wherein the biomarker is a skin anti-wrinkle related gene, and the skin anti-wrinkle related gene includes any one of 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, or a combination of at least two of them.

[0023] The combination of skin anti-wrinkle related genes provided by the present invention can be used as a biomarker for evaluating the skin anti-wrinkle ability of other raw materials or cosmetics in the future.

[0024] Preferably, the product for detecting the expression level of a biomarker comprises any one of a primer combination, a reagent or a detection model, or a combination of at least two thereof.

[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 of a series of genes provided by the present invention can be quickly used in qPCR experiments to verify the anti-wrinkle ability of new raw materials or products on the skin, and ensure the accuracy of the experimental results.

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

[0028] Table 1

[0029]

[0030]

[0031]

[0032] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

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

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

[0035] (2) The present invention provides a group 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Heat map of gene expression in the experimental and control groups. DETAILED DESCRIPTION

[0037] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0038] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0039] Example 1

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

[0041] 1. Fermentation process

[0042] First, the strain was activated, including the following steps: streaking activation on a YPD medium plate, and culturing at 30°C for 72 hours. Then, the seed solution was prepared, including the following steps: selecting a single colony with good growth in a clean bench, inoculating it into a triangular flask with YPD medium, and culturing it at 30°C and 250rpm for 30 hours. Then, the fermenter was inoculated, and the seed solution was transferred to a fermenter containing 40g / L glycerol and 4.35mL / L BSM medium.

[0043] Fermentation process control includes the following steps:

[0044] (1) Initial glycerol consumption stage: The temperature was maintained at 30°C, the pH was maintained at 5.5 with aqueous ammonia, the air flow rate was 2 vvm, and the dissolved oxygen was maintained at 20%-40% with stirring. After the dissolved oxygen value soared, the additional glycerol supplementation stage was entered.

[0045] (2) Glycerol addition stage: maintain the temperature at 30°C, use ammonia water to maintain pH at 5.5, and air flow rate at 2vvm; add 80-100mL of 50% glycerol. Observe the change rate of DO to control the flow rate, and monitor OD at all times. 600 . Waiting for OD 600 When the cell density increases to 250, stop adding glycerol and starve the cell for about 1 hour.

[0046] (3) Methanol induction stage: the temperature was controlled at 25°C and the pH was 6. 30-50 mL of pure methanol was added at a flow rate of 35 mL / h. The flow rate was determined by observing the changes in DO. Finally, the flow rate was stabilized at 10 mL of methanol per liter of culture medium per hour, based on the fact that methanol did not accumulate. Fermentation ended after 96-120 h of pure methanol induction.

[0047] 2. Purification process

[0048] (1) The obtained fermentation broth was first filtered using a 0.45 μm ceramic membrane and then subjected to a hydrophobic chromatography purification process (Tofflon hydrophobic chromatography column Phevl Purose 6FF; PrePack50 / 200). The specifications of the sample and chromatography column are shown in Table 2.

[0049] Table 2

[0050]

[0051]

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

[0053] Distilled water: 5L;

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

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

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

[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) The chromatography column was flushed with equilibration buffer at a linear flow rate of 90 cm / h for 3 CV (column volume, omitted below) until the pH, UV, and conductivity detection baselines were stable and consistent with the equilibration buffer.

[0060] 2) Load the sample and equilibration buffer at a ratio of 1:1 at a rate of 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, flush with equilibrium buffer at a speed of 90 cm / h (30 mL / min) for 2CV to wash out unbound materials until the UV detection value drops to the initial value.

[0062] 4) The elution buffer and the equilibrium buffer were eluted at a rate of 90 cm / h (30 mL / min) at a ratio of 6:4 to elute the target protein and collect the eluted fractions.

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

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

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

[0066] Example 2

[0067] In this example, transcriptomic sequencing was used to study the effect of recombinant collagen on the expression of skin anti-wrinkle related genes in Example 1. Recombinant collagen was added to the cell culture medium of human skin fibroblasts (BJ cells) growing on the wall at a final concentration of 1 mg / mL. After culturing for 24 hours, the cells were lysed with Trizon, and the cell lysate was sent to BGI for transcriptomic sequencing. The analysis scheme 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 Difference Grouping Settings

[0071] One of the important analysis points of transcriptome sequencing is the comparison of expression differences, where the experimental group is BJ cells treated with recombinant collagen, and the control group is BJ cells not treated with recombinant collagen.

[0072] 2. Experimental process

[0073] 2.1 mRNA library construction process

[0074] 1) Treat total RNA using mRNA enrichment or rRNA removal methods; mRNA enrichment: use magnetic beads with OligodT to enrich mRNA with polyA tails; rRNA removal: use DNA probes to hybridize rRNA, RNaseH selectively digests the DNA / RNA hybrid chain, and then use DNaseI to digest the DNA probe, and then obtain the desired RNA after purification.

[0075] 2) The obtained RNA was fragmented using a shearing buffer, reverse transcribed using a random N6 primer, and then the second strand of cDNA was synthesized to form double-stranded DNA.

[0076] 3) The ends of the synthesized double-stranded DNA are filled in and the 5' end is phosphorylated, forming a sticky end with a protruding "A" at the 3' end, and then a bubble-shaped linker with a protruding "T" at the 3' end is connected.

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

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

[0079] 6) Sequencing on a 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, reads with low quality, connector contamination, and high unknown base N content are filtered out. The filtered data is called clean reads. Then the clean reads are aligned to the reference genome, and then new transcript prediction, SNP&InDel and differential splicing gene detection are performed. After obtaining the new transcript, the new transcript with protein coding potential is added to the reference gene sequence to form a complete reference sequence, and then the gene expression level is calculated. Finally, for multiple samples, the differentially expressed genes between different samples are detected according to needs, and in-depth clustering analysis and functional enrichment analysis are performed on the differentially expressed genes.

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

[0084] 4.3 The FPKM values ​​of the above genes in the experimental group and the control group were integrated into the same table, and the results are shown in Table 3. The heat map analysis of the Kidio Bioinformatics Cloud tool was used to obtain the following Figure 1 Heat map shown.

[0085] Table 3

[0086]

[0087]

[0088] As can be seen from Table 3, the final calculated log2 fold change was about 0.948, that is, the gene expression was promoted by about 1.929 times, proving that recombinant collagen can increase the expression of genes related to skin anti-wrinkle.

[0089] Example 3

[0090] This example uses the recombinant collagen provided in Example 1 to verify the effect of different concentrations of recombinant collagen on the expression 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, and added in equal amounts to the cell culture medium of BJ cells growing adherently, and cultured for 24 h.

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

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

[0095] (2) Add 200 μL of chloroform to the centrifuge tube, shake the tube vigorously for 15 s, let it stand for 10 min, and then centrifuge it at 12,000 g for 15 min at 4°C.

[0096] (3) After centrifugation, the liquid was separated into layers. RNA was present in the upper colorless and transparent aqueous layer with a volume of about 600 μL. After transferring to a new centrifuge tube, 500 μL of isopropanol was added and the tube was slowly inverted several times to mix. After standing for 15 minutes, the tube was centrifuged at 12,000 g for 10 min at 4°C.

[0097] (4) After centrifugation, the RNA precipitates at the bottom of the centrifuge tube. When facing the light source, it can be seen as a gelatinous precipitate. After aspirating 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) Aspirate 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 the total volume 100 μL. Add the diluted RNA solution to the 96-well UV plate.

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

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

[0103] (4) Calculate the RNA solution concentration in μg / μL using the formula: OD260 × 0.04 × dilution factor. Dilute the RNA solution with DEPC-water to adjust the RNA concentration to 1 μg / μL.

[0104] 4. Reverse transcription

[0105] (1) Using a reverse transcription kit (Novozyme One-Step Reverse Transcription Kit R333), prepare a reverse transcription system in a 200 μL PCR tube, including: a) 1 μg / μL RNA solution, 1 μL; b) 5×qRT SuperMix, 4 μL; c) premixed enzyme solution, 1 μL; d) DEPC-water, 14 μL. The final volume of the reverse transcription system is 20 μL. Use a pipette to gently pipette and centrifuge briefly for 30 s.

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

[0107] 5. Fluorescence quantitative PCR

[0108] (1) Use the reagents in the SYBR Green fluorescent quantitative kit to prepare the reaction system in an eight-tube PCR tube, including: a) 2×SYBR Green PCR premix, volume 10 μL; b) forward and reverse primers, volume 1.5 μL each; c) cDNA solution with a concentration of 1 μg / μL, volume 1 μL; d) sterilized first-grade water, volume 6 μL. The final volume of the PCR system is 20 μL, and it is briefly centrifuged for 30 s using a handheld centrifuge.

[0109] (2) Each sample needs three replicate wells for each molecule to be tested, and a blank control without adding cDNA is 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-link PCR tube into a fluorescent quantitative PCR instrument and set the reaction program: a) 95°C for 2 min; b) 95°C for 5 min; c) 60°C for 10 s; d) 20°C constant temperature storage. Steps b and c were repeated for 45 cycles.

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

[0112] 6. Quantitative analysis of results

[0113] (1) The CT values ​​of three replicates of each sample were averaged, and the difference between the CT value of the test molecule and the CT value of the reference molecule (ACTB) was calculated, i.e., ΔCT.

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

[0115] (3) Calculate the exponential value with 2 as the base and (-ΔΔCT) as the exponent, that is, 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 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 can be seen from Table 4, after BJ cells were treated 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 values ​​of the relative expression multiples of 27 genes including COL4A1 were all greater than 1.19, indicating that the addition of recombinant collagen has the effect of promoting the expression of skin anti-wrinkle related genes. Among them, 0.5 mg / mL can ensure that the expression of all anti-wrinkle related genes is promoted and the relative expression is the highest.

[0119] Example 4

[0120] In this example, the recombinant collagen provided in Example 1 and collagen 1, collagen 2, and collagen 3 were used to measure the expression levels of skin anti-wrinkle related genes with reference to Example 3, and the measurement 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: GPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAP。

[0123] SEQ ID NO.4: GFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNT。

[0124] Table 5

[0125]

[0126]

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

[0128] Application Example 1

[0129] In this application example, the recombinant collagen provided in Example 1 is used to prepare cosmetics with anti-wrinkle function, such as facial cleansers, essences, eye creams, lotions, emulsions, facial creams or facial masks.

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

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

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

[0133] In summary, the present invention detects the expression of a series of genes related to skin anti-wrinkle by transcriptomics, proving that the recombinant collagen provided by the present invention can promote the expression of anti-wrinkle genes in skin cells. The recombinant collagen can be used for the development and application of a variety of cosmetic products with anti-wrinkle efficacy, giving cosmetic products the efficacy and effect of promoting skin anti-wrinkle ability. At the same time, the combination of skin anti-wrinkle related genes provided by the present invention can also be used as a reference for future evaluation of the anti-wrinkle ability of other raw materials or cosmetics on skin.

[0134] The applicant declares that the above is only a specific implementation mode 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 thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. The use of recombinant collagen in the preparation of a preparation for increasing the expression of genes related to skin anti-wrinkle, characterized in that: The amino acid sequence of the recombinant collagen includes the sequence shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The skin anti-wrinkle related genes include any one or a combination of at least two of collagen encoding genes, FGF family genes, TIMP family genes, GDF family genes, ELN genes, TGFB1 genes or RARA genes.

3. The use according to claim 1 or 2, characterized in that: The collagen encoding gene includes any one of COL4A1, COL6A3, COL6A6, COL7A1, COL8A1, COL11A2, COL13A1, COL16A1, COL17A1, COL23A1 or COL27A1, or a combination of at least two thereof.

4. The use according to any one of claims 1 to 3, characterized in that: The FGF family genes include any one of FGF1, FGF4, FGF5, FGF7, FGF17, FGF19, FGF21 or FGF22, or a combination of at least two of them.

5. The use according to any one of claims 1 to 4, characterized in that: The TIMP family genes include TIMP3 and / or TIMP4.

6. The use according to any one of claims 1 to 5, characterized in that: The GDF family gene includes any one of GDF5, GDF9 or GDF15 or a combination of at least two of them.

7. The use according to any one of claims 1 to 6, characterized in that: The preparation comprises a medicine and / or a cosmetic; Preferably, the cosmetics include any one of facial cleanser, essence, eye cream, toner, lotion, face cream or facial mask, or a combination of at least two thereof; Preferably, the concentration of the recombinant collagen in the preparation is 0.1-1 mg / mL.

8. Use of a product for detecting the expression level of a biomarker in evaluating the ability of cosmetics or their raw materials to increase the anti-wrinkle ability of the skin, characterized in that: The biomarker is a skin anti-wrinkle related gene, and the skin anti-wrinkle related gene includes any one or a combination of at least two of 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.

9. The use according to claim 8, characterized in that: The product for detecting the expression level of a biomarker includes any one of a primer combination, a reagent or a detection model, or a combination of at least two of them.

10. The use according to claim 9, characterized in that: The nucleic acid sequence of the primer combination includes the sequences shown in SEQ ID NO.5 to SEQ ID NO.58.

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