Recombinant collagen for promoting skin cell moisturizing capacity gene expression and application thereof

Abstract

The invention relates to recombinant collagen for promoting skin cell moisturizing capacity gene expression and application thereof. The amino acid sequence of the recombinant collagen comprises a sequence shown as SEQ ID NO.1. The invention also relates to a preparation method of the recombinant collagen. By applying the recombinant collagen provided by the invention to cells, expression of skin cell moisturizing factor generation related genes is promoted, the recombinant collagen can be used for development and application of various cosmetic products, and the cosmetic products are endowed with the efficacy and effect of promoting skin moisturizing capacity; the skin cell moisturizing factor generation related gene can also be used as a reference for evaluating the moisturizing capacity of other raw materials or cosmetics in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a recombinant collagen protein for promoting the expression of genes for the moisturizing ability of skin cells and an application thereof. Background Art

[0002] Recombinant collagen is a collagen protein that is prepared by using the amino acid sequence of collagen in the body as a template, and then rationally designed, enzymatically cleaved and spliced, and then transferred into engineered cells such as Escherichia coli and Pichia pastoris with the help of synthetic biology technology, and using 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.

[0003] CN103102407A discloses a humanized recombinant human collagen produced by eukaryotic bacteria, with a total length of 474 amino acids, composed of two identical human type III collagen fragments connected in series, and 6 histidine residues connected to the C-terminus as specific affinity purification markers. The performance of the recombinant human collagen is better than that of animal collagen and prokaryotic engineered bacteria recombinant collagen; it has a specific affinity purification marker and is easy to obtain a high-purity product.

[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, recombinant collagen has many advantages. Providing a new type of recombinant collagen and exploring its possible application scenarios have become one of the urgent issues to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a recombinant collagen protein that promotes the expression of genes for the moisturizing ability of skin cells and its application. By means of transcriptomics, the expression of a series of genes related to the production of skin moisturizing factors was detected. The recombinant collagen protein provided by the present invention can promote the expression of genes related to the production of skin cell moisturizing factors.

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

[0008] In a first aspect, the present invention provides a recombinant collagen protein for promoting the expression of a gene for the moisturizing ability of skin cells, wherein the amino acid sequence of the recombinant collagen protein includes the sequence shown in SEQ ID NO.1.

[0009] SEQ ID NO.1:GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGP AGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPP.

[0010] The present invention provides a novel recombinant collagen protein, which can promote the expression of genes related to the production of moisturizing factors in skin cells, and can be used for the development and application of various cosmetic products, and endow the cosmetic products with the efficacy and function of promoting the moisturizing ability of the skin.

[0011] Preferably, the genes promoting the expression of skin cell moisturizing ability genes include any one of ABCA1 gene, ALOX family genes, CERS family genes, CHST8 gene, CLDN3 gene, DEGS2 gene, ELOVL4 gene, FLG gene, GPR39 gene, HAS1 gene, KLK5 gene, MGAT3 gene, OVOL1 gene, PLCB4 gene, PPARG gene, SLC1A3 gene, TCHH gene or TGM gene, or a combination of at least two of them.

[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 to 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 genes related to the production of skin cell moisturizing factors. After application, the overall log2 fold change of the genes related to the production of skin cell moisturizing factors is increased by about 1.251, that is, the gene expression is promoted by about 2.38 times, which has the effect of promoting the expression of genes related to the production of skin cell moisturizing factors.

[0013] Among the genes related to the production of skin cell moisturizing factors provided by the present invention, the ABCA1 gene encodes a membrane integral protein, which helps maintain the moisture and barrier of the skin by regulating lipid metabolism in the skin; the ALOX family genes encode arachidonic acid lipoxygenase, which helps maintain the moisture and barrier of the skin by regulating lipid metabolism in the skin; the CERS family genes encode ceramide synthase, which is a key component for skin water retention and maintenance of skin barrier function. Lack of ceramide will lead to dry skin and water loss; the CHST8 gene encodes sugar sulfotransferase, which promotes the sulfation of moisturizing factor glycosaminoglycans, enhances their structural stability and hydration capacity, thereby helping to maintain the moisture balance of the skin; The 3 genes encode intercellular tight junction proteins, which help the skin retain moisture and avoid dryness by strengthening the sealing of the skin surface and preventing the evaporation of water from the external environment; the DEGS2 gene is involved in lipid metabolism, and helps maintain the structural stability and barrier function of the skin by regulating ceramide levels, maintaining the integrity and water balance of the stratum corneum; the ELOVL4 gene is involved in fatty acid biosynthesis, helping to generate important skin long-chain fatty acids, preventing water loss and maintaining the water balance of the skin; the FLG gene encodes filaggrin, which helps the skin maintain water balance by regulating keratinocyte differentiation and repair; the GPR39 gene encodes a G protein-coupled receptor, which indirectly affects the expression level of the water channel protein AQP3 by regulating the expression level of the water channel protein AQP3. The HAS1 gene encodes hyaluronan synthase, which is responsible for the production of hyaluronic acid. Hyaluronic acid has strong water absorption and can absorb water and retain it in the skin to prevent dry skin and wrinkle formation; the KLK5 gene encodes a serine protease, which is involved in the exfoliation of keratinocytes and prevents dry skin caused by excessive thickness of the stratum corneum; the MGAT3 gene encodes a glycosyltransferase, which regulates the glycosylation modification of glycoproteins, enhances the stability of glycoproteins, and promotes tight junctions between cells, thereby helping to maintain the barrier integrity and water balance of the skin; the OVOL1 gene is a key regulator of keratinocyte differentiation, which promotes keratinocytes to form a dense barrier to prevent water loss; the PLCB4 gene encodes a phospholipase, which It overregulates phospholipid signaling to maintain the moisture balance and barrier stability of the skin; the PPARG gene helps maintain skin barrier function and hydration by regulating lipid metabolism and cell differentiation; the SLC1A3 gene encodes a glutamate transporter, which contributes to the synthesis of proteins in skin cells and the differentiation of keratinocytes, and plays an important role in the formation of the skin barrier; the TCHH gene encodes a structural protein, which enhances the structure and moisture retention capacity of the skin by supporting the function of keratinocytes; the TGM gene encodes a transglutaminase, which plays an important role in the cross-linking of the extracellular matrix, and significantly affects the skin's moisturizing ability by promoting the formation of the stratum corneum, strengthening the skin barrier function, retaining the natural moisturizing factor and reducing transepidermal water loss.

[0014] Preferably, the ALOX family genes include ALOX12B and / or ALOX5.

[0015] Preferably, the CERS family genes include CERS1 and / or CERS4.

[0016] Preferably, the genes promoting the expression of skin cell moisturizing ability genes include ABCA1, ALOX12B, ALOX5, CERS1, CERS4, CHST8, CLDN3, DEGS2, ELOVL4, FLG, GPR39, HAS1, KLK5, MGAT3, OVOL1, PLCB4, PPARG, SLC1A3, TCHH and TGM.

[0017] The second invention provides the use of the recombinant collagen as described in the first aspect in the preparation of a preparation for increasing the expression of genes related to the production of moisturizing factors in skin cells.

[0018] Preferably, the genes related to skin cell moisturizing factor production include ABCA1, ALOX12B, ALOX5, CERS1, CERS4, CHST8, CLDN3, DEGS2, ELOVL4, FLG, GPR39, HAS1, KLK5, MGAT3, OVOL1, PLCB4, PPARG, SLC1A3, TCHH and TGM.

[0019] Preferably, the preparation comprises a pharmaceutical and / or a cosmetic.

[0020] 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.

[0021] 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.).

[0022] In a third aspect, the present invention provides an expression promoter for increasing the expression of genes related to skin cell moisturizing factor production, 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.

[0023] In a fourth aspect, the present invention provides the use of a product for detecting the expression level of a biomarker in evaluating the ability of cosmetics or their raw materials to increase skin moisturizing ability, wherein the biomarker is a gene related to the production of skin cell moisturizing factor, and the gene related to the production of skin cell moisturizing factor includes any one of ABCA1, ALOX12B, ALOX5, CERS1, CERS4, CHST8, CLDN3, DEGS2, ELOVL4, FLG, GPR39, HAS1, KLK5, MGAT3, OVOL1, PLCB4, PPARG, SLC1A3, TCHH or TGM, or a combination of at least two of them.

[0024] The combination of genes related to skin cell moisturizing factor production provided by the present invention can be used as a biomarker for evaluating the moisturizing ability of other raw materials or cosmetics on skin cells in the future.

[0025] 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.

[0026] Preferably, the nucleic acid sequence of the primer combination includes the sequences shown in SEQ ID NO.5 to SEQ ID NO.44.

[0027] The primers of a series of genes provided by the present invention can be quickly used in qPCR experiments to verify the moisturizing ability of new raw materials or products on the skin, and ensure the accuracy of the experimental results.

[0028] The nucleic acid sequence information of the primer combination provided by the present invention is shown in Table 1.

[0029] Table 1

[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 genes related to the production of moisturizing factors in skin cells than existing collagen proteins, and can be added to skin care products as a raw material in the future to achieve better skin care effects.

[0035] (2) The present invention provides a group of genes that promote the production of moisturizing factors in skin cells, and provides primers that can be used to detect the above genes, which can be used as a reference for evaluating the moisturizing 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 that promotes the expression of a gene that improves the moisturizing ability of skin cells. 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 pH of the effluent 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] This example uses transcriptomic sequencing to study the effect of recombinant collagen on the production of skin moisturizing factors in Example 1. Recombinant collagen is added to the cell culture medium of HaCaT cells that are growing on the wall at a final concentration of 1 mg / mL. HaCaT cells are human immortalized epidermal cells cultivated from keratinocytes. They are immortalized keratinocytes of normal human skin from non-tumor sources. They are similar to the differentiation characteristics of normal human keratinocytes and are commonly used to study the proliferation, migration and differentiation mechanisms of the skin epidermis, as well as to study the effects of active substances on keratinocytes. After 24 hours of culture, the cells were lysed with Trizon, and the cell lysate was sent to BGI for transcriptome sequencing. The analysis scheme is as follows.

[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 levels, where the experimental group is HaCaT cells treated with recombinant collagen, and the control group is HaCaT 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 genes related to skin moisturizing factor production 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] Gene name FPKM value of experimental group FPKM value of control group <![CDATA[Log2 fold change (experimental group / control group)]]> ABCA1 0.02 0.01 1 ALOX12B 0.24 0.02 3.584963 ALOX5 1.74 1.32 0.398549 CERS1 3.61 3 0.267036 CERS4 0.92 0.39 1.23816 CHST8 0.06 0 2.584963 CLDN3 0.81 0.67 0.273761 DEGS2 0.06 0.02 1.584963 ELOVL4 0.06 0 2.584963 FGR 0.02 0 1 GPR39 4.12 3.41 0.272873 HAS1 0.03 0 1.584963 KLK5 0.1 0.05 1 MGAT3 0.04 0 2 OVOL1 0.04 0 2 PLCB4 0.06 0.04 0.584963 PPARG 10.11 8.26 0.291569 SLC1A3 0.02 0 1 TCH 0.09 0.06 0.584963 TGM1 0.64 0.28 1.192645 average value 1.1395 0.8765 1.251467

[0087] As can be seen from Table 3, the final calculated log2 fold change had an overall average increase of approximately 1.251, that is, gene expression was promoted by approximately 2.38 times, proving that recombinant collagen can increase the expression of genes related to the production of moisturizing factors in skin cells.

[0088] Example 3

[0089] This example uses the recombinant collagen provided in Example 1 to verify the effect of different concentrations of recombinant collagen on the expression of genes related to skin moisturizing factor production, including the following steps:

[0090] 1. Cell sample culture

[0091] 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 HaCaT cells growing adherently, and cultured for 24 hours.

[0092] 2. Extraction and purification of total RNA in cells

[0093] (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.

[0094] (2) Add 200 μL of chloroform to the centrifuge tube, shake the tube vigorously for 15 seconds, let it stand for 10 minutes, and then centrifuge it at 12,000 g for 15 minutes at 4°C.

[0095] (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 transfer 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 minutes at 4°C.

[0096] (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.

[0097] (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.

[0098] 3. RNA concentration determination

[0099] (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 a 96-well UV plate.

[0100] (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 seconds, measure the absorbance at 260 nm and 280 nm, and read the value.

[0101] (3) Calculation of OD 260 / OD 280 Ratio: A ratio between 1.8 and 2.0 indicates that the quality of the extracted RNA meets subsequent needs.

[0102] (4) According to the formula OD 260 × 0.04 × dilution factor to calculate the RNA solution concentration in μg / μL, and dilute with DEPC-water to adjust the RNA concentration to 1 μg / μL.

[0103] 4. Reverse transcription

[0104] (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 seconds.

[0105] (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.

[0106] 5. Fluorescence quantitative PCR

[0107] (1) Using the reagents in the SYBR Green fluorescent quantitative kit, prepare the reaction system in an eight-link PCR tube, including: a) 2×SYBR Green PCR premix, volume 10μL; b) forward and reverse primers, volume 1.5μL each; c) cDNA solution with a concentration of 1μg / μL, volume 1μL; d) sterilized first-grade water, volume 6μL. The final volume of the PCR system is 20μL, and it is briefly centrifuged for 30s using a handheld centrifuge.

[0108] (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.

[0109] (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.

[0110] (4) After the PCR process is completed, the software automatically generates the CT value for each sample.

[0111] 6. Quantitative analysis of results

[0112] (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.

[0113] (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.

[0114] (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 genes related to skin cell moisturizing factor production are shown in Table 4.

[0115] Table 4

[0116]

[0117] It can be concluded from Table 4 that after treating HaCaT 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 relative expression multiples of 20 genes such as ABCA1 were all greater than 1.19, which has the effect of promoting the expression of genes related to the moisturizing ability of skin cells. When the concentration of recombinant collagen is 0.5 mg / mL, it can ensure the expression promotion of all genes related to enhancing the moisturizing ability of skin, and the average promotion rate is the highest.

[0118] Example 4

[0119] 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 genes related to skin moisturizing factor production 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.

[0120] SEQ ID NO.2:LPGYKGEPGRDGDKGDRGLPGFPGLHGMPGSKGEMGAKGDKGSPGFYGKKGAKGEKGNAGFPGLPGPAGEPGRHGKDGLMGSPGFKGEAGSPGASPGEPGYMGLPGIQGKKGDKGNQGEKGIQGQKGENGRQGIPGQQGIQGHHGAKGERGEKGEPGVRGAIGSKGESGVDGLMGPAGPKGQPGDPGPQGPPGLDGKPGRE。

[0121] SEQ ID NO.3:GPQGPKGDPGPPGIPGRNGDPGIPGQPGSPGSPGPPGICESCPTGPQNYSPQYDSYDVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAP。

[0122] SEQ ID NO.4: GFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAP GKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGG AGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETG PPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNT.

[0123] Table 5

[0124] Gene name Recombinant collagen Collagen 1 Collagen 2 Collagen 3 ABCA1 1.686 1.412 1.640 1.205 ALOX12B 9.246 3.572 5.065 5.446 ALOX5 1.323 1.118 1.215 1.056 CERS1 1.378 1.085 1.349 1.297 CERS4 2.167 1.041 1.408 1.074 CHST8 4.237 2.522 1.925 2.067 CLDN3 1.346 1.052 1.066 0.997 DEGS2 2.357 2.297 1.512 2.070 ELOVL4 4.876 4.002 1.732 1.938 FGR 1.878 1.839 1.230 0.983 GPR39 1.350 1.193 1.026 1.096 HAS1 2.351 2.018 2.134 1.323 KLK5 1.744 1.128 1.050 1.319 MGAT3 2.975 2.165 1.135 1.817 OVOL1 3.139 1.714 2.258 2.816 PLCB4 1.349 1.301 1.252 1.266 PPARG 1.135 0.959 0.983 1.018 SLC1A3 1.635 1.180 1.252 1.446 TCH 1.366 1.348 1.118 0.930 TGM1 2.139 1.207 1.877 1.667 average value 2.484 1.707 1.611 1.642

[0125] It can be seen from Table 5 that, compared with other types of collagen, the recombinant collagen provided by the present invention has the effect of promoting the expression of genes related to skin moisturizing ability, and the average promotion rate is the highest.

[0126] Application Example 1

[0127] In this application example, the recombinant collagen provided in Example 1 is used to prepare cosmetics with moisturizing function, such as facial cleanser, essence, eye cream, toner, lotion, face cream or facial mask.

[0128] 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%).

[0129] 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%).

[0130] 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%).

[0131] In summary, the present invention detects the expression of a series of genes related to the production of skin moisturizing factors by transcriptomics, proving that the recombinant collagen provided by the present invention can promote the expression of genes related to the production of skin cell moisturizing factors. The collagen can be used for the development and application of a variety of cosmetic products, giving cosmetic products the efficacy and effect of promoting the ability of skin moisturizing. At the same time, this series of genes can also be used as a reference for evaluating the moisturizing ability of other raw materials or cosmetics in the future.

[0132] 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. A recombinant collagen protein that promotes the expression of genes that enhance the moisturizing ability of skin cells, characterized in that: The amino acid sequence of the recombinant collagen includes the sequence shown in SEQ ID NO.

1.

2. The recombinant collagen according to claim 1, characterized in that The genes promoting the expression of skin cell moisturizing ability genes include any one of ABCA1 gene, ALOX family gene, CERS family gene, CHST8 gene, CLDN3 gene, DEGS2 gene, ELOVL4 gene, FLG gene, GPR39 gene, HAS1 gene, KLK5 gene, MGAT3 gene, OVOL1 gene, PLCB4 gene, PPARG gene, SLC1A3 gene, TCHH gene or TGM gene, or a combination of at least two thereof; Preferably, the ALOX family genes include ALOX12B and / or ALOX5; Preferably, the CERS family genes include CERS1 and / or CERS4.

3. The use according to claim 1 or 2, characterized in that: The genes that promote the expression of skin cell moisturizing ability include ABCA1, ALOX12B, ALOX5, CERS1, CERS4, CHST8, CLDN3, DEGS2, ELOVL4, FLG, GPR39, HAS1, KLK5, MGAT3, OVOL1, PLCB4, PPARG, SLC1A3, TCHH and TGM.

4. Use of the recombinant collagen according to any one of claims 1 to 3 in the preparation of a preparation for increasing the expression of genes related to the production of moisturizing factors in skin cells.

5. The use according to claim 4, characterized in that: The skin cell moisturizing factor production-related genes include ABCA1, ALOX12B, ALOX5, CERS1, CERS4, CHST8, CLDN3, DEGS2, ELOVL4, FLG, GPR39, HAS1, KLK5, MGAT3, OVOL1, PLCB4, PPARG, SLC1A3, TCHH and TGM.

6. The use according to claim 4 or 5, characterized in that: The preparation comprises a medicine and / or a cosmetic; 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.

7. The use according to any one of claims 4 to 6, characterized in that: 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 skin moisturizing ability, characterized in that: The biomarker is a gene related to the production of skin cell moisturizing factor, and the gene related to the production of skin cell moisturizing factor includes any one or a combination of at least two of ABCA1, ALOX12B, ALOX5, CERS1, CERS4, CHST8, CLDN3, DEGS2, ELOVL4, FLG, GPR39, HAS1, KLK5, MGAT3, OVOL1, PLCB4, PPARG, SLC1A3, TCHH or TGM.

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.44.

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