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

Abstract

The present application relates to a kind of recombinant collagen for promoting the expression of skin cell moisturizing capacity gene and its application, the amino acid sequence of the recombinant collagen includes the sequence shown in SEQ ID NO.1.By applying the recombinant collagen provided by the present application to cell, the expression of skin cell moisturizing factor production related gene is promoted, the recombinant collagen can be used in the development and application of various cosmetic products, and the cosmetic product is endowed with the efficacy and effect of promoting the moisturizing capacity of skin, and the above-mentioned skin cell moisturizing factor production related gene can also be used as the reference for later evaluation of the moisturizing capacity of other raw materials or cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to the cosmetic technology field, especially to a recombinant collagen protein for promoting the expression of skin cell moisturizing capacity genes and application thereof. BACKGROUND

[0002] The recombinant collagen protein is a collagen protein prepared by using the amino acid sequence of the collagen protein in a living body as a template, reasonably designing, enzyme cutting and splicing, and then transferring into the engineering cells such as Escherichia coli and Pichia pastoris by means of synthetic biology technology, and finally preparing by using the rapid production capacity of the engineering cells. If the recombinant collagen protein is designed and spliced by using the collagen protein of a human being as a template, it is called recombinant human collagen protein.

[0003] CN103102407A discloses a humanized gene recombinant human collagen protein produced by eukaryotic bacteria, which has a full length of 474 amino acids and is composed of two identical human type III collagen fragments connected in series, and 6 histidine residues are connected at the C terminal as a specific affinity purification marker. The gene recombinant human collagen protein has better performance than animal collagen protein and prokaryotic engineering bacteria recombinant collagen, and has a specific affinity purification marker, so that high-purity products can be easily obtained.

[0004] Compared with the animal-derived collagen prepared by a traditional method, the recombinant collagen protein has better water solubility, stronger processability, and characteristics such as single component, controllable preparation process and short production cycle, and the product quality is also easier to control. As a high polymer material with biocompatibility and biodegradability, the recombinant collagen protein has wide application prospects in the medical field, such as promoting wound healing, tissue regeneration, drug carriers and beauty and skin care, and has many benefits.

[0005] In summary, the recombinant collagen protein has many advantages, and it has become one of the problems to be solved in the present field to provide a new type of recombinant collagen protein and explore its possible application scenarios. SUMMARY

[0006] To solve the above technical problems, the present application provides a recombinant collagen protein for promoting the expression of skin cell moisturizing capacity genes and application thereof. By means of transcriptomics, the expression of a series of genes related to skin moisturizing factor production is detected, and the recombinant collagen protein provided by the present application can promote the expression of the genes related to skin moisturizing factor production.

[0007] To achieve the above purpose, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a recombinant collagen protein for promoting the expression of skin cell moisturizing capacity genes, wherein the amino acid sequence of the recombinant collagen protein comprises the sequence shown in SEQ ID NO. 1.

[0009] SEQ ID NO. 1: GPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPP.

[0010] The present application provides a new type of recombinant collagen protein, which can promote the expression of skin cell moisturizing factor generation related 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 moisturizing ability.

[0011] Preferably, the genes in the promotion of skin cell moisturizing ability gene expression include any one or a combination of at least two 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.

[0012] In bioinformatics analysis, the log2 fold change value of 0.25 is usually used as the set threshold for screening differentially expressed genes, which is converted to a relative expression multiple of about 1.19. A relative expression greater than 1.19 is considered to be promoted. The recombinant collagen protein provided by the present application can promote the expression of skin cell moisturizing factor generation related genes, and the overall log2 fold change of the skin cell moisturizing factor generation related genes after application is about 1.251, i.e. the gene expression is promoted by about 2.38 times, which has the effect of promoting the expression of skin cell moisturizing factor generation related genes.

[0013] Among the skin cell moisturizing factor generation-related genes provided by the present application, the ABCA1 gene encodes a membrane-integrated protein that helps maintain skin moisture and barrier function by regulating lipid metabolism in the skin; the ALOX family gene encodes arachidonic acid lipoxygenase, which helps maintain skin moisture and barrier function by regulating lipid metabolism in the skin; the CERS family gene encodes ceramide synthase, and ceramide is a key component of skin water retention and maintenance of skin barrier function, and a lack of ceramide can cause dry skin and water loss; the CHST8 gene encodes a sugar sulfotransferase that enhances the structural stability and water-binding capacity of moisturizing factor glycosaminoglycans by promoting their sulfation, thereby helping to maintain skin moisture balance; the CLDN3 gene encodes an intercellular tight junction protein that helps skin retain moisture and avoid dryness by strengthening the sealing of the skin surface to prevent evaporation of moisture in the external environment; the DEGS2 gene is involved in lipid metabolism, which helps maintain the structural stability and barrier function of the skin by regulating ceramide levels, maintains the integrity of the stratum corneum and water balance; the ELOVL4 gene is involved in fatty acid biosynthesis, which helps generate important skin long-chain fatty acids to prevent water loss and maintain skin moisture balance; the FLG gene encodes filaggrin, which helps maintain skin moisture balance by regulating keratinocyte differentiation and repair; the GPR39 gene encodes a G protein-coupled receptor that indirectly affects the skin's moisturizing ability by regulating the expression level of water channel protein AQP3; the HAS1 gene encodes hyaluronic acid synthase, which is responsible for the generation of hyaluronic acid, which has strong water absorption and can absorb water and retain it in the skin, preventing dry skin and wrinkle formation; the KLK5 gene encodes a serine protease that is involved in the exfoliation of keratinocytes, preventing dry skin caused by excessive thickness of the stratum corneum; the MGAT3 gene encodes a glycosyltransferase that enhances the stability of glycoproteins by regulating their glycosylation modification, promotes intercellular tight junctions, and thus helps maintain the barrier integrity and water balance of the skin; the OVOL1 gene is a key regulator of keratinocyte differentiation that promotes the formation of a dense barrier to prevent water loss; the PLCB4 gene encodes a phospholipase that maintains skin moisture balance and barrier stability by regulating phospholipid signaling; the PPARG gene helps maintain skin barrier function and hydration by regulating lipid metabolism and cell differentiation; the SLC1A3 gene encodes a glutamate transporter that helps synthesize proteins in skin cells and differentiate keratinocytes, playing an important role in the formation of the skin barrier; the TCHH gene encodes a structural protein that enhances the structure and moisture retention capacity of the skin by supporting the function of keratinocytes; and the TGM gene encodes transglutaminase, which plays an important role in the cross-linking of the extracellular matrix, significantly affecting the skin's moisturizing ability by promoting the formation of the stratum corneum, strengthening the skin barrier function, retaining natural moisturizing factors, and reducing transdermal water loss.

[0014] Preferably, the ALOX family gene comprises ALOX12B and / or ALOX5.

[0015] Preferably, the CERS family gene comprises CERS1 and / or CERS4.

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

[0017] The second aspect, the present application provides the use of the recombinant collagen protein as described in the first aspect in the preparation of a preparation for increasing the expression of a skin cell moisturizing factor production related gene.

[0018] Preferably, the skin cell moisturizing factor production related gene comprises 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 drug and / or a cosmetic.

[0020] Preferably, the cosmetic comprises any one or a combination of at least two of facial cleanser, essence, eye cream, toner, emulsion, cream or mask.

[0021] Preferably, the concentration of the recombinant collagen protein 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] The third aspect, the present application provides an expression promoter for increasing the expression of a skin cell moisturizing factor production related gene, wherein the expression promoter is a recombinant collagen protein, and the amino acid sequence of the recombinant collagen protein comprises the sequence shown in SEQ ID NO. 1.

[0023] In a fourth aspect, the present application provides use of a product for detecting expression level of a biomarker, which is a skin cell moisturizing factor production related gene, including 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, in evaluating moisturizing ability of a cosmetic or a raw material thereof on skin.

[0024] The present application provides a combination of skin cell moisturizing factor production related genes as biomarkers for evaluating moisturizing ability of other raw materials or cosmetics on skin in the future.

[0025] Preferably, the product for detecting expression level of a biomarker includes any one or a combination of at least two of a primer combination, a reagent or a detection model.

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

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

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

[0029] Table 1

[0030]

[0031]

[0032] Other specific point values in the above numerical ranges can be selected, which will not be repeated here.

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

[0034] (1) The present application provides a new recombinant collagen protein, which has better ability to promote expression of skin cell moisturizing factor production 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 effect.

[0035] (2) The present application provides a group of genes that promote skin cell moisturizing factor production, and provides primers that can be used to detect the above genes, which can be used as a reference for evaluating moisturizing 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 promotes the expression of genes related to skin cell moisturizing ability. The amino acid sequence of the recombinant collagen is shown in SEQ ID NO.1. The preparation method of this 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 phase: control temperature is 25°C, pH is 6, and pure methanol is added at a flow rate of 35 mL / h for 30-50 mL; the flow rate is determined by observing the DO change; finally, the flow rate is stabilized at 10 mL of methanol per liter of culture medium per hour, and the fermentation is completed after 96-120 hours of pure methanol induction.

[0047] 2. Purification process

[0048] (1) The obtained fermentation broth is first filtered using a 0.45 μm ceramic membrane, and then subjected to a hydrophobic chromatography purification process (Dongfenglong hydrophobic chromatography column Phevl Purose 6FF; PrePack 50 / 200), and the sample and chromatography column specifications are shown in Table 2.

[0049] Table 2

[0050]

[0051]

[0052] (2) Experimental material preparation and buffer preparation:

[0053] Distilled water: 5 L;

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

[0057] The above buffers must be filtered with a 0.45 μm filter membrane.

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

[0059] 1) The chromatography column is 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 are stable and consistent with the equilibration buffer.

[0060] 2) The sample and equilibration buffer are loaded at a ratio of 1:1 at a speed of 60 cm / h (20 mL / min), and the flow-through components during loading are collected and detected for the purity and content of the target protein.

[0061] 3) After loading is completed, the equilibration buffer is flushed at a speed of 90 cm / h (30 mL / min) for 2 CV to wash out the unbound materials until the UV detection value decreases to the initial value.

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

[0063] 5) Wash the chromatography column with at least 2 column volumes of 1M NaOH at a speed 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 chromatography column with 5-10 CV of distilled water at a speed of 60 cm / h (20 mL / min) to flush to neutral pH and close to 0 conductivity of the effluent to remove 1M NaOH.

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

[0066] Example 2

[0067] This example uses transcriptome sequencing to study the effect of recombinant collagen on skin moisturizing factor production in Example 1. Recombinant collagen is added to the cell culture medium of HaCaT cells growing in adherence at a final concentration of 1 mg / mL. HaCaT cells are human immortalized epidermal cells derived from keratinocytes, which are non-tumor-derived human normal skin immortalized keratinocyte strains similar to normal human keratinocyte differentiation characteristics, commonly used to study the proliferation, migration and differentiation mechanisms of skin epidermis, and to study the effects of active substances on keratinocytes. After 24 hours of culture, the cells are lysed with Trizon, and the cell lysate is sent to Huada Gene for transcriptome sequencing. The analysis scheme is shown below.

[0068] 1. Analysis scheme

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

[0070] 1.2 Differential group settings

[0071] One of the important analysis points of transcriptome sequencing is the differential comparison of expression levels, with the experimental group being HaCaT cells treated with recombinant collagen and the control group being HaCaT cells not treated with recombinant collagen.

[0072] 2. Experimental process

[0073] 2.1 mRNA library construction flow

[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.

[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 moisturizing factor generation-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] Gene name Experimental group FPKM value Control group FPKM value Log2 fold change (experimental / control) 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 TCHH 0.09 0.06 0.584963 TGM1 0.64 0.28 1.192645 Average value 1.1395 0.8765 1.251467

[0087] From Table 3, it can be seen that the final calculation of log2 fold change is overall average promotion of about 1.251, that is, the gene expression is promoted by about 2.38 times, which proves that the recombinant collagen can promote the expression amount of skin moisturizing factor generation related genes.

[0088] Example 3

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

[0090] 1. Cell sample culture

[0091] The collagen was configured into 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and 1.0 mg / mL concentrations of test solutions, which were respectively added into the cell culture medium of the adherent growing HaCaT cells in equal amounts, and cultured for 24 h.

[0092] 2. Total RNA extraction and purification in cells

[0093] (1) The culture medium was removed from the cultured cells, washed twice with pre-cooled PBS, 1 mL of TRIzol reagent was added, and the collected centrifuge tube was blown and collected, and placed at room temperature for 5 min.

[0094] (2) 200 μL of chloroform was added to the centrifuge tube, the centrifuge tube was vigorously shaken for 15 s, and after standing for 10 min, it was centrifuged at 4°C at 12000 g for 15 min.

[0095] (3) After centrifugation, the liquid was layered, and the RNA was in the upper colorless transparent aqueous phase layer with a volume of about 600 μL. After transferring to a new centrifuge tube, 500 μL of isopropanol was added, mixed slowly by inverting several times, and after standing for 15 min, it was centrifuged at 4°C at 12000 g for 10 min.

[0096] (4) After centrifugation, the RNA precipitate was at the bottom of the centrifuge tube, and a gelatinous precipitate was visible when facing the light source. After removing the supernatant, 1 mL of 75% ethanol was added to resuspend and wash the RNA, and it was centrifuged at 4°C at 7500 g for 5 min.

[0097] (5) The ethanol supernatant was removed, the RNA was dried for 5 min, and 20 μL of DEPC-water was added to dissolve the RNA.

[0098] 3. RNA concentration determination

[0099] (1) Take 1 μL of 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 ultraviolet version.

[0100] (2) Place the 96-well version in a full-wavelength enzyme marker, select the area to be tested, and set the program to: shake for 10 s, measure the 260 nm and 280 nm absorbance values, and read the values.

[0101] (3) Calculate OD 260 / OD 280 The ratio, which is between 1.8 and 2.0, indicates that the quality of the extracted RNA meets the subsequent needs.

[0102] (4) According to the formula OD 260 × 0.04 × dilution factor, the concentration of the RNA solution is calculated, which is μg / μL. Adjust the RNA concentration to 1 μg / μL with DEPC-water.

[0103] 4. Reverse transcription

[0104] (1) Use the reverse transcription kit (Novozyme one-step reverse transcription kit R333) to prepare the reverse transcription system in a 200 μL PCR tube, which contains: a) 1 μg / μL of 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 mix gently, and use a portable centrifuge for short centrifugation for 30 s.

[0105] (2) Place the reverse transcription system PCR tube in the RCR instrument, set the reaction program as: a) incubate at 50℃ for 15 min; b) inactivate at 85℃ for 10 s, the mRNA in the system is reverse transcribed into cDNA.

[0106] 5. Fluorescent quantitative PCR

[0107] (1) Use the reagents in the SYBR Green fluorescent quantitative kit to prepare the reaction system in an eight-tube PCR tube, which contains: a) 2×SYBR Green PCR premix, 10 μL; b) forward and reverse primers, 1.5 μL each; c) 1 μg / μL of cDNA solution, 1 μL; d) sterilized primary water, 6 μL. The final volume of the PCR system is 20 μL, use a portable centrifuge for short centrifugation for 30 s.

[0108] (2) Each sample needs three repeated holes for each molecule to be tested, and a blank control without adding cDNA is set. In addition to the molecules to be tested, actin beta (ACTB) is also needed as a reference molecule.

[0109] (3) Put the eight PCR tubes into the fluorescence quantitative PCR instrument, and set the reaction program: a) 95℃ for 2 min; b) 95℃ for 5 min; c) 60℃ for 10 s; d) 20℃ constant temperature storage. The steps b and c are repeated for 45 cycles.

[0110] (4) After the end of the PCR program, the software automatically generates the CT value of each sample.

[0111] 6. Quantitative analysis of results

[0112] (1) Take the average of the CT values of each sample in triplicate, and calculate the difference between the CT value of the tested molecule and the CT value of the reference molecule (ACTB), that is, ΔCT.

[0113] (2) Calculate the difference between the ΔCT value of the tested molecule in the cells treated with the raw material and the ΔCT value of the tested molecule in the untreated cells, that is, ΔΔCT.

[0114] (3) Calculate the exponential value with 2 as the base and (-ΔΔCT) as the exponent, that is, 2 -△△CT Take the average of 2 -△△CT of each concentration of parallel groups as the quantitative result of the tested molecule in each sample, realize the quantitative detection at the gene level, and the test results of skin cell moisturizing factor generation related genes are shown in Table 4.

[0115] Table 4

[0116]

[0117] From Table 4, it can be concluded that after treating HaCaT cells with 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL and 1.0 mg / mL concentrations of recombinant collagen, the average relative expression fold of 20 genes such as ABCA1 is 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 promotion of the expression of all genes related to the enhancement of skin moisturizing ability, 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 determine the expression of skin moisturizing factor generation related genes according to Example 3, and the determination 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: GFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNT.

[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 TCHH 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] As can be seen from Table 5, the recombinant collagen protein provided by the present application has an effect of promoting expression of a gene related to skin moisturizing ability, and the average promotion rate is the highest compared to other types of collagen proteins.

[0126] Application Example 1

[0127] In this application example, the recombinant collagen protein provided in Example 1 is applied to the preparation of a cosmetic having a moisturizing function, which can be, for example, a facial cleanser, serum, eye cream, toner, emulsion, cream, or pack.

[0128] Application 1, preparation of a cream, including the following components in mass percentage: 0.1% recombinant collagen protein, 7% caprylic / capric acid triglyceride, 5% liquid paraffin, 4% glycerol, 3% propylene glycol, 3% ethylhexyl palmitate, 3% vaseline, 2% ceteareth-21, 2% dimethicone, 2% cetearyl alcohol, 1.5% ceteareth-2, 1.5% glyceryl monostearate, 0.2% methyl paraben, 0.2% carbomer, 0.2% triethanolamine, 0.1% ethyl paraben, and the balance being water (65.2%).

[0129] Application 2, preparation of an emulsion, including the following components in mass percentage: 0.1% recombinant collagen, 5% glycerin, 5% hydrogenated polyisobutene, 4% ethylhexyl palmitate, 3% propylene glycol, 3% caprylic / capric triglyceride, 2% dimethicone, 1.5% cetostearyl alcohol ether-21, 1.2% cetostearyl alcohol ether-2, 0.5% tocopherol, 0.2% nipagin methyl ester, 0.1% triethanolamine, 0.1% nipagin ethyl ester, 0.1% carbomer, the rest being water (74.2%).

[0130] Application 3, preparation of a cosmetic water, including the following components in mass percentage: 0.1% recombinant collagen, 5% glycerin, 3% propylene glycol, 0.2% diazimidazolidinyl urea, the rest being water (91.7%).

[0131] In summary, the present application detects the expression of a series of genes related to the generation of skin moisturizing factors by transcriptomics, proving that the recombinant collagen provided by the present application can promote the expression of skin cell moisturizing factor generation related genes. The collagen can be used for the development and application of various cosmetic products, giving the cosmetic products the efficacy and effect of promoting skin moisturizing ability. At the same time, the 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 states that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by any person skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A recombinant collagen protein that promotes the expression of a gene for the moisturizing ability of skin cells, characterized by, The amino acid sequence of the recombinant collagen is shown as SEQ ID NO.

1.

2. Use of the recombinant collagen according to claim 1 in the preparation of a cosmetic product for increasing the expression amount of a gene related to the generation of a skin cell moisturizing factor.

3. Use according to claim 2, characterized in that, The cosmetic product is any one or a combination of at least two of a facial cleanser, an essence, an eye cream, a cosmetic water, a lotion, a cream, or a mask.

4. Use according to any one of claims 2-3, characterized in that, The concentration of the recombinant collagen in the cosmetic product is 0.1-1 mg / mL.

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