A small molecule multifunctional information peptide and its application

Abstract

The present invention provides a small molecule multifunctional information peptide and its application, belonging to the technical field of active peptide raw material development. The small molecule multifunctional information peptide sequence provided by the present invention is VHPYVPR, which can effectively bind to the downstream receptor of EGF, simulate the function of EGF, and avoid the situation that the existing cell growth factor raw materials are limited in use in the cosmetics field due to molecular weight problems. Experiments have shown that the small molecule multifunctional information peptide provided by the present invention can promote the repair of skin damage such as scratches, inhibit the release of inflammatory factors, inhibit the activity of β-galactosidase, and promote the production of type I, type III and type XVII collagen, showing excellent repair, soothing and anti-aging functions, and strong permeability, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of active peptide raw material development, in particular to a small molecule multifunctional information peptide and application thereof. Background Art

[0002] Epidermal Growth Factor (EGF) is a type of polypeptide substance widely found in mammals. It was first discovered by American scientist Dr. Cohen. Due to his outstanding contribution to the study of EGF, Dr. Cohen was awarded the Nobel Prize in Medicine and Physiology in 1986. EGFR (Epidermal Growth Factor Receptor) is the receptor for EGF cell proliferation and signal transduction.

[0003] Human epidermal growth factor (hEGF) is a small molecule polypeptide composed of 53 amino acids, with a molecular weight of about 6000 Daltons and an isoelectric point of about 4.6. There are three pairs of disulfide bonds in the molecule, so it is relatively stable to physical and chemical factors such as acid, alkali, and heat. hEGF has a wide range of biological effects and can strongly promote the growth of various epidermal tissues. It has been used in medicine to treat burns, ulcers, various types of trauma, and corneal injuries. hEGF can also promote the metabolism of normal epidermal cells. Adding it to transdermal care products can achieve whitening, repair, anti-wrinkle, and anti-aging effects. However, due to its large molecular weight, transdermal absorption is more difficult.

[0004] However, in general cosmetic technology and application fields, hEGF is restricted in use in the cosmetic field due to its large molecular weight, difficulty in being absorbed by the skin, and the risk of causing unpredictable risks, as well as regulatory restrictions.

[0005] Small molecule peptides have received extensive attention in the fields of beauty, skin care, anti-aging and functional foods in recent years due to their efficient absorption, regulatory repair ability, and anti-aging and anti-inflammatory effects. However, the current peptide synthesis technology is mainly based on chemical synthesis, which has problems such as high cost, low yield, and difficulty in impurity control. Phage display technology is a powerful and widely used method in drug development, protein-protein interaction research and antibody engineering. The present invention selects heptapeptides as peptide segments for screening targets, mainly based on the fact that heptapeptides can show sufficient structural diversity in a shorter amino acid sequence, which can not only ensure efficient binding with the target, but also avoid folding and non-specific binding that may be produced by longer peptide chains. Plant homologous peptides do not contain common allergens in animal proteins, and are used in transdermal care products for sensitive skin and atopic dermatitis populations, with higher safety. Secondly, the molecular weight of plant-derived peptides is generally 200-3000 Da, which is easily absorbed through the skin, combined with skin cells or deep into the skin, and exerts significant effects. It is easier to be used by the skin than traditional macromolecular ingredients, which improves the effectiveness of the efficacy concentration. Finally, plant-derived peptides have diverse functions and comprehensive effects. For example, plant-derived peptides are rich in antioxidant amino acids (such as tyrosine and tryptophan), which can scavenge free radicals, delay skin aging, and reduce the damage of oxidative stress to the skin, and have antioxidant effects; some plant-derived peptides can effectively relieve skin sensitivity by regulating skin inflammatory factors, such as reducing the levels of IL-6 and TNF-α, and have anti-inflammatory and soothing effects. Plant-derived peptides contain highly hydrophilic amino acid sequences that can enhance the skin's hydration capacity, while promoting skin barrier repair, and have moisturizing and repairing effects. Plant homologous peptides are safe and mild, have good biocompatibility and multiple biological activities. The diversity of plant-derived peptides provides broad resources and possibilities for the screening of multifunctional information peptides.

[0006] Chinese patent CN101469021A discloses the development of a recombinant human epidermal growth factor (hEGF) and interleukin (IL-23), in which hEGF and IL-23 cDNAs were synthesized by RT-PCR, purified, amplified, and digested, and then cloned into the eukaryotic expression vector pcDNA3.1(+). The expression product of the recombinant plasmid is hEGF-IL-23 fusion protein, which has a role in the healing of chronic ulcers after burns and anti-infection immune protection, but the scheme does not achieve the improvement of hEGF permeability.

[0007] The prior art still lacks small molecule functional active peptide raw materials with similar or even better cosmetic effects than hEGF and good permeability. Summary of the invention

[0008] The purpose of the present invention is to provide a small molecule multifunctional information peptide and its application, which has excellent repairing, soothing and anti-aging functions and strong permeability.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides an EGF-like peptide, the amino acid sequence of the EGF-like peptide is shown as SEQ ID NO.1.

[0011] The present invention also provides a gene encoding the above-mentioned EGF-like peptide, and the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0012] The present invention also provides a bacteriophage, which is a bacteriophage displaying the above-mentioned EGF-like peptide.

[0013] The present invention also provides an expression vector, which comprises an initial vector and the above gene.

[0014] The present invention also provides the use of the above-mentioned EGF-like peptide, gene, phage or expression vector in the preparation of transdermal care preparations.

[0015] The present invention also provides the use of the above-mentioned EGF-like peptide, gene, phage or expression vector in the preparation of any of the following products:

[0016] ① Products that promote the proliferation of immortalized keratinocytes;

[0017] ② Products that promote the migration of human immortalized keratinocytes;

[0018] ③Filaggrin or laminin expression products;

[0019] ④β-galactosidase activity inhibitor;

[0020] ⑤ Products that inhibit inflammatory factors;

[0021] ⑥Products that promote type I collagen gene expression;

[0022] ⑦Products that promote the expression of type III collagen gene;

[0023] ⑧Products that promote the expression of type XVII collagen gene.

[0024] The present invention also provides a cosmetic containing the EGF-like peptide.

[0025] Preferably, the cosmetic is a repair product, a soothing product or an anti-aging product.

[0026] Preferably, the concentration of the EGF-like peptide in the cosmetic is above 1 ppm.

[0027] The present invention also provides a medical product, wherein the medical product contains the above-mentioned EGF-like peptide.

[0028] Beneficial effects of the present invention:

[0029] The small molecule peptide raw material provided by the present invention, the EGF-like peptide, is obtained by screening the phage random display seven peptide library, and shows a high degree of homology with the polypeptide of a certain type of plant in nature. The peptide has a small molecular weight of only 867 Da, high safety, low risk of allergies, can effectively bind to the downstream receptor of EGF, simulate the function of EGF, mild effect and diverse functions, thereby avoiding the situation that the existing cell growth factor raw materials are limited in the field of cosmetics due to molecular weight problems. Experiments have shown that the EGF-like peptide provided by the present invention can promote the repair of skin damage phenomena such as scratches as an active peptide raw material, inhibit the release of inflammatory factors, inhibit the activity of β-galactosidase, show excellent repair, soothing, anti-aging functions, and strong permeability, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of ELISA color development for positive clones;

[0031] Figure 2 is the effect of C2642 on the proliferation of HaCaT cells;

[0032] Figure 3 Data graph showing C2642 promoting migration of HaCaT cells;

[0033] Figure 4 Figure 2 shows C2642 promoting the migration of HaCaT cells;

[0034] Figure 5 Promote C2642 FLG Genes and LN Gene expression profile;

[0035] Figure 6 This is a photograph of the results of the fluorescent staining test to determine the activity of β-galactosidase;

[0036] Figure 7 This is a statistical analysis result diagram of the percentage of β-galactosidase staining area;

[0037] Figure 8 Promote C2642 COL1A1 Gene expression profile;

[0038] Fig. 9 Promote C2642 COL3A1 Gene expression profile;

[0039] Fig.10 Promote C2642 COL17A1 Gene expression profile;

[0040] Fig.11This is a comparative test result chart for inhibiting soothing-related genes;

[0041] Fig.12 This is the result of the human body Raman test of the permeability of C2642;

[0042] Fig.13 This is the result of the Raman test on the permeability of EGF in the human body. DETAILED DESCRIPTION

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] Example 1

[0045] Phage panning and titer determination of peptide surrogate antigens

[0046] (1) Coating: Prepare a 10 μg / mL monoclonal antibody EGFR solution in PBS and coat 8 wells of a 96-well ELISA plate with 100 μL per well at 4°C overnight. To remove nonspecific adsorption, take another 8 wells and coat them with 300 μL of 10 μg / mL BSA / PBS overnight.

[0047] (2) Blocking: The next day, discard the supernatant from the ELISA plate, wash the plate three times with 1% PBST solution, take 100 μL of the freshly amplified phage peptide library and add it to the wells coated with BSA. Block the other 8 wells with 200 μL of 3% skimmed milk powder and incubate at 37°C for 1 h.

[0048] (3) First round of screening: discard the blocking solution and wash the plate three times. Transfer the supernatant of the phage peptide library after the reaction to the wells coated with EGFR, shake at room temperature for 2 h, discard the supernatant, and wash the plate 10 times with 1% PBST solution; then, add 100 μL of elution solution, shake at room temperature for 10 min, add neutralizing solution, adjust the pH value to between 7-8, and collect.

[0049] (4) Determine the titer of the eluted phage, infect Escherichia coli ER2738 that has grown to the early logarithmic phase, amplify the phage, determine the titer of the amplified phage, and use it for the second round of selection.

[0050] (5) Select a single colony of ER2738 and place it in 5 mL of LB medium. Cultivate it at 37°C with shaking for 4-8 h until the logarithmic phase (OD 600 =0.5).

[0051] (6) Preheat the LB / IPTG / Xgal plate in a 37°C incubator 1 hour in advance.

[0052] (7) Use LB medium to dilute the phage to different concentrations.

[0053] (8) Aliquot the ER2738 bacterial suspension that has been amplified to the logarithmic phase into EP tubes, 200 μL / tube. Add 10 μL of the diluted phage to the bacterial suspension, vortex, and leave at room temperature for 1-5 min.

[0054] (9) Pour the phage-infected bacterial solution onto a preheated LB / IPTG / Xgal plate, spread it evenly, and invert it in a 37°C constant temperature incubator for overnight culture.

[0055] (10) After 24 h of culture, the original phage titer was calculated based on the number of blue plaques on the plate.

[0056] Phage random display seven peptide library was used to pan EGF alternative antigen. After three rounds of bio-panning, the changes in the phage titer obtained from each round of panning were measured. The results showed that phages that bind to EGFR were present in each round of panning, that is, phage peptides that can bind to EGFR antibodies were enriched. The phage titers of each round of panning during phage screening are shown in Table 1.

[0057] Table 1 Phage titer in each round of panning

[0058]

[0059] Amplification and identification of phage display peptide libraries

[0060] (1) Take out Escherichia coli ER2738 from the -80℃ ultra-low temperature freezer, use an inoculation loop to take a small amount of bacterial liquid and streak it on an LB-tetracycline plate, and culture it in a constant temperature incubator at 37℃ overnight.

[0061] (2) The next day, pick a single colony from the LB-tetracycline plate, inoculate it into 20 mL of culture medium, and culture it in a 250 mL culture bottle at 37°C and 250 rpm until the OD of the culture solution reaches 0. 600 It is 0.01-0.05.

[0062] (3) Take an appropriate amount of phage display peptide library and add it to the ER2738 bacterial culture that has been cultured to the early logarithmic phase, and culture it at 37°C with shaking for 4.5 h.

[0063] (4) Transfer the bacterial solution to a centrifuge tube and centrifuge at 12,000 g and 4°C for 10 min.

[0064] (5) Transfer the supernatant to a clean centrifuge tube and add 1 / 4 volume of 20% PEG / 2.5 M NaCl to precipitate the phages. Mix well and incubate on ice for 2 h.

[0065] (6) Centrifuge at 12,000 rpm and 4°C for 15 min, discard the supernatant, and after a brief centrifugation, remove the remaining supernatant with a micropipette.

[0066] (7) Add 1 mL of PBS solution to dissolve the precipitate, centrifuge the phage solution at maximum speed, and transfer the supernatant to a clean centrifuge tube for later use.

[0067] (8) Pick a single ER2738 clone in 2 mL LB medium and culture it at 37°C overnight with shaking. The next day, take 50 μL of the overnight bacteria and dilute it 1:100 into 5 mL LB medium. Randomly pick 20 single colonies from the fresh third-round panning eluate phage titer plate and place them in 5 mL of bacterial solution. Culture them at 37°C with shaking for 4.5 h. After centrifugation at 14,000 rpm for 30 s, take the supernatant for phage ELISA experiment.

[0068] (9) Prepare 10 μg / mL EGFR antibody solution with PBS buffer and coat the 96-well ELISA plate. Prepare 3% BSA-PBS solution to coat the ELISA plate, 100 μL per well. Each phage clone corresponds to a group of ELISA wells coated with antibodies and a group of wells coated with BSA. Coat overnight at 4°C. The next day, discard the supernatant and wash the plate three times. Block each well with 3% skim milk powder and block at 37°C for 1 h. Wash the plate three times, add 100 μL phage supernatant, incubate at 37°C for 0.5 h, discard the supernatant, wash the plate 10 times, add 100 μL 1:10000 diluted M13-HRP, react at 37°C for 45 min, wash the plate 6 times, add freshly prepared substrate colorimetric solution, develop at 37°C for 15 min, and finally add 50 μL stop solution to terminate the reaction. At the same time, use an ELISA reader to measure the absorbance at 450 nm. The schematic diagram of ELISA colorimetric of positive clones is shown below. Figure 1 shown.

[0069] Table 2 Absorbance values ​​of positive clones at 450 nm

[0070]

[0071] like Figure 1 As shown in FIG. 2 , the wells that did not develop color with BSA but developed color with EGFR were positive clones, and a total of 6 positive clones were screened. As shown in Table 2 , C2642 had the strongest binding affinity with EGFR.

[0072] Sequence analysis and synthesis of phage positive clones

[0073] The culture medium of Escherichia coli infected by the positive phage clone was sent to the sequencing center for sequence analysis. The sequencing primer was 96 gⅢ (5´-CCCTCATAGTTAGCGTAACG-3´, such as SEQ ID NO.3).

[0074] Table 3 Gene sequences and amino acid sequences of phage positive clones

[0075]

[0076] The monoclones with large differences in OD values ​​were sequenced, and the results are shown in Table 3. A phage-positive clone amino acid sequence was screened out, VHPYVPR (SEQ ID NO.1), and by comparing the peptide database, it was found that it showed high homology with the peptides of a certain type of plant in nature. According to the amino acid sequence, the sequence was modified to the backbone from C-terminus to N-terminus, and the DNA sequence corresponding to the amino acid sequence was introduced into the yeast fermentation system using a seamless cleavage site. After expression, the broken products were collected, and then purified by high-performance liquid phase and freeze-dried to obtain the heptapeptide-53 peptide (denoted as C2642) for subsequent efficacy testing.

[0077] Experimental Example 1 Human immortalized keratinocytes (HaCaT) activity experiment

[0078] 1. Prepare culture medium containing C2642

[0079] The polypeptide lyophilized powder obtained in Example 1 and the commercially available EGF standard were first diluted to 1000 ppm using Dulbecco's Modified Eagle Medium (DMEM), and filtered using a 0.22 μM sterile filter membrane. The solution was then gradiently diluted to 100 ppm, 50 ppm, 10 ppm and 5 ppm to obtain the test samples.

[0080] 2. Determination of cell viability

[0081] Take cells in logarithmic growth phase and calculate 8*10 3 100 μL of the prepared sample solution was added to each well and incubated for 24 h. After adding 10 μL of CCK-8 reagent and incubating for 1 h, the OD value was measured at 450 nm using an enzyme reader. The results are as follows: Figure 2 shown.

[0082] The results showed that compared with the blank control group, the cell survival rates of the experimental groups added with C2642 and EGF were all greater than 100%, and the above four concentrations of C2642 could promote the proliferation of HaCaT. C2642 reached 117% at a concentration of only 5 ppm. This result shows that the C2642 of the present invention has a significant effect on promoting the proliferation of HaCaT cells.

[0083] Experimental Example 2: Promoting Cell Migration Experiment

[0084] Human immortalized keratinocytes in good growth condition were cultured at 1*10 6The cells were seeded in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. After culturing for 24 h, use a 10 μL pipette tip to measure a ruler and draw two vertical cross lines (+ shape), wash the cells three times with PBS, and remove the scratched cells. Then add 2 mL of samples of different concentrations to each well. The serum-free DMEM medium was used as the control group. The sample groups were added with 10 ppm and 50 ppm doses of C2642 solution and commercially available EGF standard solution, respectively, to stimulate the cells for 24 h. Observe and take pictures under an inverted microscope at 0 h and 24 h after sample loading. Use Image J software for data analysis to calculate the scratch area value and cell migration rate. Figure 3 , Figure 4 shown.

[0085] The results showed that HaCaT cells treated with 50 ppm C2642 or the positive control EGF standard at the same concentration grew well and stretched and spread on the scratched surface ( Figure 4 ). These results indicate that the EGF-like peptide (C2642) screened by phage screening has biological activity similar to that of EGF and can promote cell migration.

[0086] Experimental Example 3 C2642 regulates the expression of filaggrin and laminin genes

[0087] 1. HaCaT cells in logarithmic growth phase were divided into 1*10 6 pcs / well were inoculated in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 24 h;

[0088] 2. Discard the culture medium and treat with H2O2 solution (150 μM) for 1.5 h (the blank control group does not need H2O2 solution treatment);

[0089] 3. The experimental group was added with samples of various concentrations, and the blank control group and negative control group were added with serum-free culture medium, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0090] 4. Discard the supernatant, wash twice with PBS, and collect the cells;

[0091] 5. Extract RNA, reverse transcribe it into cDNA, and then perform RT-qPCR to detect the filaggrin gene ( FLG )Laminin ( LN )'s relative expression level;

[0092] 6. Use EXCEL software to analyze the data. The results are as follows: Figure 5 shown.

[0093] FLGThe protein encoded by the gene is filaggrin, which is an important protein in epidermal keratinocytes and is involved in the formation and maintenance of the stratum corneum. It promotes the formation of the stratum corneum by binding to keratin fibers, thereby exerting the physical barrier function of the skin. LN The protein encoded by the gene is laminin, which is one of the important components of the extracellular matrix. Laminin is mainly present in the basement membrane area of ​​the epidermis and dermis. It maintains the stability and structural integrity of the basement membrane by interacting with integrins, heparan sulfate proteoglycans, etc. At the same time, it can promote the adhesion of keratinocytes to the basement membrane, thereby enhancing the mechanical strength of the skin barrier.

[0094] The results showed that compared with the control group, treatment with 50 ppm C2642 or the same concentration of positive control EGF standard could promote FLG and LN Gene expression, FLG The relative gene expression levels increased by 56.04% and 70.51% respectively; LN The relative expression of genes increased by 36.56% and 31.37%, respectively. The above results show that the EGF-like peptide (C2642) screened by phage has biological activity similar to EGF and can promote the expression of filaggrin and laminin with barrier repair function.

[0095] Experimental Example 4 Anti-aging efficacy comparison test

[0096] β-Galactosidase activity assay

[0097] 1. Take cells with good growth status and add 5×10 5 The cells were inoculated at a density of 1000 cells / mL in a 6-well culture plate and cultured overnight at 37°C in a 5% CO2 environment.

[0098] 2. Remove the waste culture medium from the cell culture plate that has been cultured overnight, add hydrogen peroxide stimulation, and then add corresponding sample treatments to the cells according to the sample groups in the experimental example 1. Set up 3 replicates for each treatment, and incubate for 24 hours after addition.

[0099] 3. Remove the cell treatment medium from step 3, wash once with PBS, add 1 mL of staining fixative, and fix for 15 minutes at room temperature. Aspirate the fixative, wash the cells 3 times with PBS, 3 min each time. Aspirate all PBS, add 1 mL of SA-β-gal staining solution to each well. Incubate at 37℃ overnight, cover with plastic wrap to prevent evaporation of the staining solution, and then observe and take pictures under an optical microscope. The results are as follows: Figure 6 shown.

[0100] Cellular senescence is a persistent state of cell cycle arrest that accumulates in aging organisms, leading to tissue dysfunction and driving the appearance of aging-related phenotypes. During the cellular senescence process, a major feature is the increased activity of lysosomal β-galactosidase, which is also called senescence-associated β-galactosidase (SA-β-gal). SA-β-gal is a widely used marker for identifying senescence in different types of senescent cells and in vivo and in vitro experiments.

[0101] Comparative test experiments have shown that C2642 is significantly better than EGF peptide in inhibiting β-galactosidase activity and alleviating aging. Figure 7 As shown in the figure, the blue-green substance (β-galactosidase) in the fibroblasts of the model group treated with hydrogen peroxide increased, indicating that the cells were in a state of senescence. C2642 and EGF peptide were added to the senescent cells respectively, and the reduction of blue-green substances in samples treated with different concentrations reflected the alleviation of senescence by C2642 and EGF peptide.

[0102] like Figure 6 and Figure 7 As shown in the figure, compared with the control group, after hydrogen peroxide stimulation, the green substance in the model group increased. As the concentration of added samples C2642 and EGF peptide increased, the cell β-galactosidase activity decreased, and the green substance in the figure decreased, indicating that C2642 and EGF peptide can inhibit the activity of β-galactosidase and alleviate cell aging. Statistical analysis of the proportion of β-galactosidase staining area showed that Figure 7 ,like Figure 7 As shown in the figure, the percentage of β-galactosidase staining area in the C2642 treatment group at different concentrations was lower than that in the EGF peptide treatment group. In the figure, the percentage of β-galactosidase staining area in the experimental group treated with C2642 at a concentration of 50 ppm was the smallest, which was different from the model group, and the inhibition rate reached 67.21%. In contrast, the EGF peptide treatment group was able to reduce the activity of β-galactosidase, but its effect was not as good as that of the C2642 treatment group.

[0103] Therefore, based on all the concentration results, C2642 can better inhibit the activity of β-galactosidase in cells and slow down the aging process of cells in vivo compared with EGF peptide, that is, C2642 is more effective in delaying cell aging than EGF peptide.

[0104] C2642 regulates collagen expression assay

[0105] 1. HaCaT cells in logarithmic growth phase were divided into 1*10 6pcs / well were inoculated in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 24 h;

[0106] 2. Discard the culture medium and treat with H2O2 solution (150 μM) for 1.5 h (the blank control group does not need H2O2 solution treatment);

[0107] 3. The experimental group was added with samples of various concentrations, and the blank control group and negative control group were added with serum-free culture medium, and cultured in a 37°C, 5% CO2 incubator for 24 h;

[0108] 4. Discard the supernatant, wash twice with PBS, and collect the cells;

[0109] 5. Extract RNA, reverse transcribe it into cDNA, and then perform RT-qPCR to detect type I collagen gene ( COL1A1 )、Type III collagen gene( COL3A1 ) and type XVII collagen gene ( COL17A1 )'s relative expression level;

[0110] 6. Use EXCEL software to analyze the data. The results are as follows: Figure 8 , Fig. 9 , Fig.10 shown.

[0111] Reactive oxygen stimulation can cause tissue oxidative stress and inflammation, which in turn leads to reduced collagen synthesis, resulting in loose and inelastic skin, and the skin condition gradually deteriorates. Type I collagen (Collagen Ⅰ) is a major structural protein and the main component of the dermis of the skin. It provides basic support and tension for the skin, keeping the skin firm and elastic. However, with age, the synthesis of type I collagen gradually decreases, resulting in thinning of the dermis, decreased elasticity, wrinkles, sagging skin and other signs of aging. Type III collagen (Collagen Ⅲ) has a wide distribution range and is high in the skin, which can maintain the elasticity and firmness of the skin. The regeneration capacity of type III collagen in the human body is limited. After adulthood, the synthesis of type III collagen in the fibroblasts of the human body's own dermis will gradually decrease. The reduced expression of type XVII collagen (Collagen ⅩVII) may lead to reduced adhesion between epidermal stem cells (ESC) and the basement membrane, and then affect the stemness and proliferation ability of ESC, causing changes in the thickness and stratification of the aging epidermis. Type XVII collagen can also promote the proliferation and renewal of epidermal stem cells, promote competitive regeneration of skin tissue, and delay aging.

[0112] like Figure 8 As shown in the figure, compared with the blank control group, the expression of type I collagen (Collagen I, COL1A1) gene relative expression decreased by 42.34%. Compared with the model group, after 24 h of exposure to 5 ppm and 50 ppm samples, COL1A1 The relative expression of genes increased significantly by 70.07% and 112.19% respectively. Compared with the model group, the 5 ppm and 50 ppm samples of EGF peptide had a significant effect after 24 hours. COL1A1 The relative gene expressions increased by 61.17% and 70.83%, respectively.

[0113] like Fig. 9 Compared with the blank control group, the COL3A1 The relative expression of the gene decreased by 26.20%. COL3A1 The relative gene expression level increased significantly by 52.73%. Compared with the model group, the 5 ppm and 50 ppm samples of EGF peptide had a significant effect on the expression of genes after 24 hours. COL3A1 The relative gene expressions increased by 27.68% and 39.83%, respectively.

[0114] like Fig.10 Compared with the blank control group, the COL17A1 Compared with the model group, 5 ppm and 50 ppm C2642 respectively COL17A1 The relative expression of genes increased significantly by 47.85% and 72.94%. Compared with the model group, the 5 ppm and 50 ppm samples of EGF peptide had an effect on the expression of genes after 24 hours. COL17A1 The relative gene expressions increased by 10.97% and 52.35%, respectively.

[0115] Comparative test experiments have shown that C2642 is significantly better than EGF peptide in promoting the gene expression of type I collagen, type III collagen and type XVII collagen. Therefore, compared with EGF peptide, C2642 is more effective in slowing down the aging process of cells in the body, that is, C2642 is better than EGF peptide in delaying cell aging.

[0116] Experimental Example 5: Soothing effect comparison test

[0117] C2642 regulates inflammatory factor-related gene determination experiment

[0118] 1. Prepare culture medium containing C2642

[0119] The polypeptide lyophilized powder obtained in Example 1 and the commercially available EGF standard were first diluted to 1000 ppm using Dulbecco's Modified Eagle Medium (DMEM), and filtered using a 0.22 μM sterile filter membrane. The solution was then gradiently diluted to 50 ppm and 5 ppm to obtain the test samples.

[0120] 2. Preparation of HaCaT cells

[0121] Take cells in good growth state and place 5×10 5 The cells were inoculated at a density of 1000 cells / mL in a 6-well culture plate and cultured overnight at 37°C in a 5% CO2 environment.

[0122] 3. Treat cells

[0123] The waste culture medium in the cell culture plate cultured overnight was removed, and after adding hydrogen peroxide stimulation, the cells were added with corresponding sample treatments according to the sample groups in the experimental example 1, with 3 replicates for each treatment, and incubated for 24 h after addition.

[0124] 4. RT-qPCR determination to explore the expression of soothing-related target genes

[0125] The culture medium of the above cells was removed, the cells were collected, lysate was added, total RNA of the cells was extracted, RNA concentration and purity were detected, and collagen-related genes were determined by RT-qPCR technology. TNF-α (tumor necrosis factor-α), IL-6 The relative expression level of interleukin-6 gene.

[0126] Comparative test experiments have shown that C2642 has a significantly better inhibitory effect on soothing-related genes than EGF peptide. Fig.11 As shown in the figure, the model group treated with hydrogen peroxide showed a difference from the blank control group, indicating that the cells were in an inflammatory state after stimulation. After adding C2642 and EGF peptide to the cells in the inflammatory state, the upregulation of inflammatory factor gene expression caused by external stimulation was improved, indicating that C2642 can inhibit inflammatory factors and achieve the effect of soothing the skin.

[0127] like Fig.11 As shown in the figure, with the increase of the concentration of added sample C2642 and EGF peptide, the gene expression of IL-6 and TNF-α inflammatory factors in the cells was downregulated. IL-6 After gene expression analysis, compared with the model group, 5 ppm and 50 ppm C2642 peptide treatments were able to IL-6The relative expression of genes was down-regulated by 45.70% and 59.83% after treatment with 5 ppm and 50 ppm EGF peptide, respectively. IL-6 Gene expression was downregulated by 7.01% and 19.11%. TNF-α After gene expression analysis, compared with the model group, 5 ppm and 50 ppm C2642 treatments could respectively TNF-α The relative expression of genes was down-regulated by 27.73% and 54.59%; after treatment with 50ppm EGF peptide, TNF-α The relative expression of the gene was downregulated by 24.69%.

[0128] Therefore, based on the results of all soothing-related gene tests, C2642 has a stronger ability to inhibit the expression of inflammatory factor genes than EGF peptide, that is, C2642 is more effective in soothing the skin than EGF peptide.

[0129] Experimental Example 6 C2642 percutaneous permeability test

[0130] 1. Select the front area of ​​the human forearm for human Raman test: the samples used for the test are C2642 and EGF, and the application amount is 5 mg / cm 2 , select 1×1 cm 2 The LabRAM Odyssey high-speed and high-resolution confocal Raman spectrometer (HORIBA) was used to conduct the test at 1 h, 2 h, 4 h, 6 h, and 8 h; the test number was 1 person, and the test area was 3 1×1 cm 2 The skin area tested was 5 points in each test area, and the test was repeated 3 times in parallel. The relative permeability of C2642 and EGF was calculated.

[0131] 2. Raman spectroscopy imaging data processing includes spectral preprocessing and data analysis. The spectral preprocessing process includes cosmic ray removal, spectral smoothing, background noise removal, baseline calibration and spectral normalization. Univariate data analysis mainly analyzes the Raman spectral data of the biochemical substance corresponding to a specific peak position to show the distribution of the substance in human skin.

[0132] 3. Data analysis: Labspec software was used to perform baseline calibration of the Raman spectrum and confirm the position of characteristic peaks, and to calculate the obtained Raman spectrum, including peak intensity, peak shift, peak area, half-peak width, etc.; at the same time, Labspec software was used to perform numerical analysis of the peak intensity corresponding to different depths and to draw its spatial distribution.

[0133] 4. After the obtained Raman spectrum is preprocessed, the initial position of the Raman signal is used as the 0 μm position on the skin surface. After feature selection, the keratin / lipid related characteristic information is selected to determine the junction of the stratum corneum and the active epidermis and the junction of the active epidermis and the dermis according to the change of the depth in the skin and the change of the moisture content in the skin, so as to confirm the information related to the skin depth. The characteristic Raman signal of the product, which is different from the intrinsic signal of the skin, is used to confirm its distribution in different skin depth spaces, so as to describe the permeability behavior of the product. The data normal distribution test (Shapiro-Wilk Test) is performed using ORIGIN 2017 software. If it is normally distributed, a paired t Otherwise, the two-sample rank sum test is used.

[0134] 5. Relative permeability (%) = Raman spectrum intensity of active substance entering the skin / Raman spectrum intensity of active substance applied to the skin * 100%

[0135] like Fig.12 As shown in the figure, after using C2642 on human skin, C2642 penetrated into the stratum corneum within 0.5 h and broke through the stratum corneum in small amounts; within 2 h, C2642 continued to penetrate into the stratum corneum and the active epidermis; within 4-6 h, C2642 continued to penetrate into the stratum corneum and the active epidermis. The relative permeability within 0.5 h, 2 h, 4 h and 6 h was 1.87%, 3.96%, 9.21% and 12.33% respectively.

[0136] like Fig.13 As shown in the figure, after using EGF peptide on human skin, EGF peptide penetrated into the stratum corneum in small amounts within 2 h, 4 h and 6 h. The relative permeability within 0.5 h, 2 h, 4 h and 6 h was 0%, 1.13%, 2.67% and 2.38%, respectively. The results showed that C2642 screened by phage had stronger permeability than EGF peptide.

[0137] Experimental Example 7 Safety Test

[0138] C2642 Multiple Skin Irritation Test

[0139] 1. Multiple skin irritation test on New Zealand white rabbits

[0140] The C2642 obtained in Example 1 was sent to Shanghai Microspectra Testing Technology Group Co., Ltd. for multiple (acute) skin irritation tests to test its irritation. Experimental animals: New Zealand white rabbits (female); weight: 2.203kg-2.326kg. Experimental animal use license number: SYXK (Shanghai) 2021-0023. The experimental results are shown in Table 4 below:

[0141] Table 4 Results of multiple skin irritation tests on New Zealand white rabbits

[0142]

[0143] Note: The average score of each animal per day (skin irritation index) = the total score of erythema and edema of each animal for 14 days / (number of tested animals × number of days of skin irritation test)

[0144] From the results of multiple skin irritation tests in Table 4, it can be seen that the multiple skin irritation of C2642 to New Zealand white rabbits is: no irritation. Therefore, the results of multiple skin irritation tests of the multifunctional peptide C2642 provided by the present invention show that there is no skin irritation.

[0145] The C2642 obtained in Example 1 was sent to Shanghai Microspectra Testing Technology Group Co., Ltd. for acute eye irritation. Experimental animals: New Zealand white rabbits (female); body weight: 2.016kg-2.241kg. Experimental animal use license number: SYXK (Shanghai) 2021-0023. The experimental results are shown in Table 5 below:

[0146] Table 5 Results of acute eye irritation test on New Zealand white rabbits

[0147] (Test conditions: no flushing)

[0148]

[0149] Note: The integral mean is rounded to 2 decimal places.

[0150] From the acute eye irritation test results in Table 5, it can be seen that the acute eye irritation of C2642 provided by the present invention to New Zealand white rabbits is: no irritation, which proves that it is non-irritating and has broad application prospects in the preparation of cosmetics and even medical dressings and other products.

[0151] It can be seen from the embodiments of the present invention that the multifunctional signal peptide provided by the present invention is obtained by screening the phage random display seven-peptide library, which shows a high homology with the polypeptide of a certain type of plant in nature, has high safety, low allergy risk, a small peptide molecular weight, can effectively bind to the EGF receptor, has excellent repairing, soothing and anti-aging functions, and has strong permeability, mild effect, and good safety performance.

[0152] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An EGF-like peptide, characterized in that The amino acid sequence of the EGF-like peptide is shown in SEQ ID NO.

1.

2. The gene encoding the EGF-like peptide according to claim 1, characterized in that The nucleotide sequence of the gene is shown as SEQ ID NO.

2.

3. A bacteriophage, characterized in that: The phage is a phage displaying the EGF-like peptide according to claim 1.

4. An expression vector, characterized in that: The expression vector comprises an initial vector and the gene according to claim 2.

5. Use of the EGF-like peptide according to claim 1, the gene according to claim 2, the bacteriophage according to claim 3 or the expression vector according to claim 4 in the preparation of a transdermal care preparation.

6. Use of the EGF-like peptide according to claim 1, the gene according to claim 2, the bacteriophage according to claim 3 or the expression vector according to claim 4 in the preparation of any of the following products: ① Products that promote the proliferation of immortalized keratinocytes; ② Products that promote the migration of human immortalized keratinocytes; ③Filaggrin or laminin expression products; ④β-galactosidase activity inhibitor; ⑤ Products that inhibit inflammatory factors; ⑥Products that promote type I collagen gene expression; ⑦Products that promote the expression of type III collagen gene; ⑧Products that promote the expression of type XVII collagen gene.

7. A cosmetic, characterized in that: The cosmetic contains the EGF-like peptide according to claim 1.

8. The cosmetic according to claim 7, characterized in that: The cosmetics are repair products, soothing products or anti-aging products.

9. The cosmetic according to claim 7, characterized in that: The concentration of the EGF-like peptide in the cosmetic is above 1 ppm.

10. A medical product, characterized in that: The medical product contains the EGF-like peptide according to claim 1.

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