Silk polyprotein mimetic peptide and application thereof

Abstract

The invention relates to a silk protein mimetic peptide and application thereof. The amino acid sequence of the silk protein mimetic peptide is SEQ ID NO. 1. The sequence contains citrulline which is sensitive to relevant enzymes for degradation of the sericin, can be easily recognized and degraded by degrading enzymes in a dry environment, can improve the water-retaining property of epidermis, has the effect of a natural moisturizing factor, can relieve the problem of dry skin caused by lack of the sericin, can be used for cosmetics, and has a relatively good moisturizing effect. Compared with an in-vitro expression silk protein monomer, the mimic peptide has the advantages of smaller molecular weight, easiness in synthesis, low cost and better application value.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and particularly relates to a filaggrin mimetic peptide and application thereof. Background Art

[0002] Filaggrin (FLG) is an important protein in the process of epidermal keratin formation. It is obtained by shearing and modification of the precursor protein "profilaggrin". Human profilaggrin is composed of 10-12 FLG repeating units, which are cleaved and modified by specific proteases to form mature FLG monomers. Each FLG monomer contains about 324 amino acid residues with high content of histidine, arginine, serine and glycine. These amino acids not only provide filaggrin with the ability to bind to keratin fibers, but also provide raw materials for the skin's moisturizing factor (NMF). Common ichthyosis caused by filaggrin mutations is one of the most common skin diseases, characterized by dry and scaly skin, and ichthyosis is the most important risk factor for atopic dermatitis (AD). About 50% of AD patients carry FLG loss-of-function mutations, which manifest as skin itching, desquamation, thickening, erythematous papules, exudation, etc. The defective surface barrier formation caused by the loss of filaggrin and the reduced water retention are important causes of common ichthyosis and AD. The cross-linking process of filaggrin mainly involves the special cell differentiation process of cornification. Cornification is the process of final differentiation and death of epidermal cells (keratinocytes), forming the outermost layer of the skin, the stratum corneum, which provides a physical and permeability barrier for the skin. Cornification begins when keratinocytes migrate from the basal layer to the spinous layer. These cells lose their ability to divide and begin to synthesize structural proteins and enzymes related to cornification. The cross-linking of filaggrin occurs in the granular layer, forming an insoluble protein structure, the cornified envelope. This envelope replaces the cell's plasma membrane and provides a scaffold for lipid attachment. In addition to filaggrin, the proteins involved in cornification include keratin, endokeratin, exokeratin, and small proline-rich proteins. In the skin, TGM1, TGM3, and TGM5 are the main transglutaminases involved in the formation of the cornified envelope. TGM2 exists outside the cell and its expression increases when the skin is damaged. It plays an important role in the connection between cornified cells.

[0003] As the cells move outward, only the first few layers of the stratum corneum, the inner layers, contain filaggrin, while the outer layers retain keratin and filaggrin is degraded. The process of filaggrin degradation includes, after completing its function of promoting the collapse of keratin filaments, the conversion of positively charged arginine residues to citrulline by peptide arginine deiminases (PAD1, PAD3) in the granular layer, followed by further degradation into peptides of varying masses by caspase 14, calpain 1, and furin, and finally degradation into free amino acids by bleomycin hydrolase (BMH). The final mixture of amino acids, as well as trans-urocanic acid produced by histidine deamination, 2-pyrrolidonecarboxylic acid (PCA) produced by glutamine cyclization, and other metabolites, is known as natural moisturizing factor (NMF), which helps maintain the moisture of the stratum corneum by attracting water into corneocytes, which is essential for the barrier function of the stratum corneum. Patients carrying loss-of-function FLG mutations show greatly reduced levels of NMF. During the degradation of filaggrin, calpain 1 has a high activity towards deiminated amino acids, and BMH also has citrulline aminopeptidase activity, so the deimination of FLG is crucial for its degradation.

[0004] At present, there are studies on supplements for the deficiency of filaggrin. Some studies have synthesized filaggrin in vitro through eukaryotic expression, and used it for in vitro application after purification. However, the cost of synthesis and purification is high, and the molecular weight of filaggrin is large, which is not conducive to its penetration into the stratum corneum after application. In addition, the first step to catalyze the degradation of FLG requires the deimination of arginine to form citrulline, while the site of peptide arginine deiminase is located in the deeper granular layer under the stratum corneum of the epidermis, which is not conducive to the recognition and degradation of filaggrin applied on the surface into NMF by the degradation enzyme. Therefore, it is of great significance to explore a filaggrin-mimicking peptide with a small molecular weight that mimics its degradation function. Summary of the invention

[0005] In view of the above problems, the present application provides a filaggrin mimetic peptide having the effect of alleviating dry skin caused by filaggrin deficiency and its application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A filaggrin mimetic peptide, the amino acid sequence of which is SEQ ID NO.1.

[0008] Application of the filaggrin mimetic peptide in preparations for relieving dry skin and in cosmetics.

[0009] Beneficial Effects of the Invention

[0010] 1. The filaggrin mimetic peptide of the present invention contains citrulline which is sensitive to enzymes related to filaggrin degradation and is easily recognized and degraded by degradation enzymes in a dry environment, thereby improving the water retention of the epidermis. The filaggrin mimetic peptide has the effect of being degraded into a natural moisturizing factor (NMF).

[0011] 2. The filaggrin mimetic peptide of the present invention has the effect of alleviating the problem of dry skin caused by filaggrin deficiency.

[0012] 3. Compared with the in vitro expressed filaggrin monomer, the filaggrin mimetic peptide of the present invention has a smaller molecular weight, is easier to synthesize, and has a low cost.

[0013] 4. The filaggrin mimetic peptide of the present invention can be applied to cosmetics, such as moisturizing lotion, essence, facial mask or facial cream, etc., which can relieve dry skin and has moisturizing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The amino acid composition of the filaggrin mimetic peptide of the present invention is compared with the amino acid composition of human natural filaggrin;

[0015] Figure 2 It is the high performance liquid chromatography of the filaggrin mimetic peptide of the present invention;

[0016] Figure 3 The effect of the filaggrin mimetic peptide of the present invention on the activity of fibroblasts;

[0017] Figure 4 The effect of the filaggrin mimetic peptide of the present invention on the water content of the stratum corneum of the filaggrin gene knockout mouse model;

[0018] Figure 5 The results of HE staining of tissue sections showing the effect of the filaggrin mimetic peptide of the present invention on the epidermis of a filaggrin gene knockout mouse model;

[0019] Figure 6 The effect of the filaggrin mimetic peptide of the present invention on the epidermal thickness of the filaggrin gene knockout mouse model;

[0020] Figure 7 The scanning electron microscopic results of the effects of the filaggrin mimetic peptide of the present invention on the epidermis of the filaggrin gene knockout mouse model;

[0021] Figure 8 The WB test results of the filaggrin mimetic peptide of the present invention as a substrate for bleomycin hydrolase; Fig. 9 The invention discloses the effect of the skin care cream containing the filaggrin mimetic peptide of the present invention on the water content of the stratum corneum of the filaggrin gene knockout mouse model. DETAILED DESCRIPTION

[0022] In order to more clearly describe the technical solution of the present invention, the following is a detailed description through examples. The reagents and equipment used in the examples are conventional reagents and equipment unless otherwise specified, and the experimental conditions not specified are based on conventional experimental conditions.

[0023] Example 1. Synthesis and screening of filaggrin mimetic peptides

[0024] The amino acid sequence of human natural filaggrin was compared with that of mouse, rat and other species to find the conserved regions. The amino acid sequence similarity between human and mouse filaggrin was 27.5%, between human and rat filaggrin was 28.7%, and between human and pig filaggrin was 27.6%. Since the filaggrin linker sequence will be cut by a series of enzyme steps during the release from the filaggrin precursor, the subsequent moisturizing function of the mature filaggrin monomer should not involve the role of the linker sequence (1-14aa). Other conserved sequences are scattered in the filaggrin amino acid sequence, among which there are relatively concentrated similar sequences near the 30th, 80th, 150th, 160th, 210th, 230th, 250th, and 280th amino acids. During the degradation process, filaggrin is cleaved by the furin recognition sequence RRSRR (SEQ ID NO.3) (i.e., amino acids 152-156) and the caspase 14 recognition sequence VSQDSDSEGHSED (SEQ ID NO.4) (i.e., amino acids 161-173), respectively. Therefore, it is inferred that the smaller peptide fragments after cleavage should be outside the recognition regions of these two enzyme cleavage sites.

[0025] After comparison, multiple peptides with similar amino acids between different species were selected in order to find peptides that can better simulate the partial functions of filaggrin. The selected peptides are:

[0026] 31-46aa, GRQGSRHEQAQDSSRH; (SEQ ID NO.5)

[0027] 48-64aa, ASQYGQDTIRGHPGSSR; (SEQ ID NO.6)

[0028] 86-105aa,HSHTTSQGRSDASRGQSGSR; (SEQ ID NO.7)

[0029] 134-149aa,HADISRHSQAVQGQSE; (SEQ ID NO.8)

[0030] 175-190aa,ERWSGSASRNHRGSVQ; (SEQ ID NO.9)

[0031] 210-225aa, HGHSADRSRQSGTRHA; (SEQ ID NO.10)

[0032] 237-252aa, HEQARSSPGERHGSRH; (SEQ ID NO. 11)

[0033] 252-268aa,HQQSADSSRHSGIPRGQ; (SEQ ID NO.12)

[0034] 278-293aa,HWGSSGSQASDSEGHS; (SEQ ID NO.13)

[0035] 308-323aa,GPHQQSHQESARDRSG. (SEQ ID NO.14)

[0036] The amino acid sequence of human filaggrin (324aa) is as follows:

[0037] TSGHSGSFLYQVSTHEQSESSHGWTGPSTRGRQGSRHEQAQDSSRHSASQYGQDTIRGHPGSSRGGRQGYHHEHSVDSSGHSGSHHSHTTSQGRSDASRGQSGSRSASRTTRNEEQSGDSSRHSVSRHHEASTHADISRHSQAVQGQSEGSRRSRRQGSSVS QDSDSEGHSEDSERWSGSASRNHRGSVQEQSRHGSRHPRSHHEDRAGHGHSADRSRQSGTRHAETSSGGQAASSHEQARSSPGERHGSRHQQSADSSRHSGIPRGQASSAVRDSRHWGSSGSQASDSEGHSEESDTQSVSGHGQAGPHQQSHQESARDRSGG. (SEQ ID NO.2)

[0038] PeptideRanker was used to predict the biological activities of each peptide segment. The top three polypeptide sequences in descending order of biological activity were GPHQQSHQESARDRSG (SEQ ID NO.14) (0.26826), HQQSADSSRHSGIPRGQ (SEQ ID NO.12) (0.22044), and ASQYGQDTIRGHPGSSR (SEQ ID NO.6) (0.208769).

[0039] ToxinPred was used to predict toxicity, and the results showed that all sequences were non-toxic; AllerCatPro was used to predict sensitization, and all peptides were non-sensitizing. After comparison, the differences in the repeating sequences of several filaggrin monomers in the filaggrin precursor were found, and the aspartic acid D in the GPHQQSHQESARDRSG (SEQ ID NO.14) sequence was adjusted to glycine G, which increased its biological activity to 0.33129. Finally, the composition of the three filaggrin mimetic peptides screened was compared with the amino acids of natural filaggrin, and it was found that the amino acid composition of GPHQQSHQESARGRSG (SEQ ID NO.15) was similar to that of natural filaggrin. In the other two polypeptides, the proportion of amino acid I in HQQSADSSRH SGIPRGQ (SEQ ID NO.12) was 6.75 times that of I in human filaggrin, and the proportion of amino acids I and Y in ASQYGQDTIRGHPGSSR (SEQ ID NO.6) was 6.75 times that of I and Y in human filaggrin, which was quite different. Therefore, GPHQQSHQESARGRSG (SEQ ID NO. 15) was selected for subsequent verification.

[0040] Serine, glycine, alanine, proline, and glutamic acid in filaggrin all have good hydrophilicity, which can help retain skin moisture and enhance the skin's barrier function. 2-Pyrrolidonecarboxylic acid (PCA) produced by glutamine cyclization is a component of NMF. Due to its moisturizing properties, it plays an important role in maintaining skin hydration and skin barrier. Histidine is metabolized to trans-urocanic acid by histidase, which plays a role in retaining water and maintaining the acid-base balance of the skin. Arginine is deiminated by arginase to form citrulline, which can not only participate in hydration, but also improve the recognition of peptides by various degradative enzymes, promoting the orderly degradation of filaggrin. Therefore, the amino acid ratio in filaggrin is closely related to its water-retaining function, and polypeptides with amino acid sequences close to those of natural filaggrin are more likely to have the function of simulating filaggrin.

[0041] The polypeptide obtained after screening is GPHQQSHQESARGRSG (SEQ ID NO.15), in which the proportions of amino acids R, H, S, E, G, and A are similar to those of human filaggrin. Figure 1 .

[0042] The R-arginine in the polypeptide GPHQQSHQESARGRSG (SEQ ID NO. 15) was replaced with Cit-citrulline to simulate its deiminated state and improve its recognition efficiency by degradation enzymes such as calpain 1 and BMH.

[0043] The amino acid sequence of the finally obtained filaggrin mimetic peptide is: (SEQ ID NO.1)

[0044] GPHQQSHQESA{Cit}G{Cit}SG, composed of 16 amino acids (Cit is represented by a single letter X in the sequence listing).

[0045] If three letters are used, it is represented as:

[0046] Gly Pro His Gln Gln Ser His Gln Glu SerAib Cit Gly Cit Ser Gly.

[0047] The sequence can be synthesized by conventional solid phase synthesis.

[0048] The HPLC analysis of the filaggrin mimetic peptide SEQ ID NO.1 is as follows: Figure 2 The results showed that under the detection wavelength of 220nm, the target peak appeared at 6.225min, indicating that the obtained filaggrin mimetic peptide had a high purity. The peak area was calculated and it was found that the purity of the filaggrin mimetic peptide reached 95.015%.

[0049] Example 2. Effect of filaggrin mimetic peptide on fibroblast viability

[0050] The CCK8 method was used to detect the effect of the filaggrin mimetic peptide on fibroblast viability to evaluate its safety. The CCK-8 reagent contains a water-soluble tetrazolium salt (WST-8), which can be reduced to an orange tetrazolium salt product by dehydrogenases in the cell when the cell metabolic activity is present. When the cell survival or proliferation ability is strong, more WST-8 will be reduced to produce the corresponding yellow substance. After the reaction, the absorbance (OD value) of the reaction solution is measured at a specific wavelength (usually 450nm) by an enzyme reader, which is proportional to the viability of the cell.

[0051] The specific steps are as follows: 5000 fibroblasts are inoculated into each well of a 96-well plate, 100 μL / well, and when the cells are fused to 70%, different concentrations of the above-mentioned filaggrin mimetic peptide (0, 1, 2, 4, 8 and 16 mg / ml) are added, and incubated for 24 hours. 10 μL of CCK-8 reagent is added to each well, and incubated for 2 hours. The absorbance (OD value) of each well is measured at a wavelength of 450 nm using an ELISA reader. The cell viability is calculated as follows:

[0052] Cell viability = OD value of experimental group / OD value of control group × 100% = [(As-Ab) / (Ac-Ab)] × 100%;

[0053] That is, the cell viability is the ratio of the OD value of the experimental group with added filaggrin to the OD value of the control group without added filaggrin, and the OD values ​​of each group are the ratios after excluding the interference of cell culture medium and CCK8 solution.

[0054] Where As: OD value of the experimental well (containing cells, culture medium, CCK-8 solution and filaggrin mimetic peptide)

[0055] Ab: OD value of blank well (containing culture medium, CCK-8 solution, without cells and filaggrin mimetic peptide)

[0056] Ac: OD value of control well (containing cells, culture medium, CCK-8 solution, but not filaggrin mimetic peptide).

[0057] Test results such as Figure 3 As shown. Figure 3 It can be seen that when the concentration of the filaggrin mimetic peptide is greater than 2 mg / ml, the cell viability gradually increases with the increase of concentration, which indicates that the filaggrin mimetic peptide promotes the proliferation of human fibroblasts at a certain concentration without obvious cytotoxicity.

[0058] Example 3. Performance test of the filaggrin mimetic peptide of the present invention

[0059] In order to verify the moisturizing effect of the obtained filaggrin mimetic peptide, filaggrin knockout heterozygous FLG+ / - mice were selected as a filaggrin deficiency model for testing.

[0060] The FLG+ / - mice and wild-type mice used in the examples were all male, weighing 20-25 g, and purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The experiment was divided into three groups, wild-type WT mice, FLG+ / - group mice, and a filaggrin mimetic peptide treatment group, i.e., SEQ ID NO.1 FLG+ / - group. They were raised under ambient temperature (22±1°C), humidity (50±10%), and 12h light / dark cycle conditions.

[0061] After dissolving 20 mg of the filaggrin mimetic peptide of the present invention in 5 ml of deionized water, 5 ml of 40% glycerol was added, and the pH was adjusted to about 5.5 to obtain a 2 mg / ml filaggrin mimetic peptide solution that was easy to apply. The solution was stored at 4° C. for later use.

[0062] After the mice were adapted to the feeding environment for 7 days, they were anesthetized and the hair on the back of the neck was shaved, with the shaved area being about 1 cm 2 The FLG+ / - group mice were smeared with 20% glycerol deionized water solution on the shaved area as a control; the SEQ ID NO.1 FLG+ / - group was smeared with 20% glycerol deionized water solution with 2 mg / ml filaggrin mimetic peptide added on the shaved area. The application was done twice a day, and the skin tissue of the mice was taken for HE staining and scanning electron microscopy observation after 7 days.

[0063] 1. Effect of filaggrin mimetic peptide on the water content of the stratum corneum

[0064] Six hours after the last application of the filaggrin mimetic peptide, the mice were anesthetized and a non-invasive skin detector (Courage & Khazak, Germany) was used. The CM825 probe detects the moisture content of the stratum corneum of the skin. During the test, press the probe vertically on the treated area of ​​the mouse, press the probe lightly, lift it up, let the probe rebound, and read the result.

[0065] The results are as follows Figure 4 As shown. Figure 4 It can be seen that the water content of the stratum corneum of the skin of the FLG+ / - group mice is significantly lower than that of the wild-type WT group mice. Since the FLG+ / - group mice are heterozygous for filaggrin knockout, their filaggrin content is lower than that of normal wild-type WT mice, resulting in a decrease in their epidermal water retention capacity and a decrease in the water content of the stratum corneum. The water content of the stratum corneum of the skin of the FLG+ / - group mice treated with the filaggrin mimetic peptide, i.e., SEQ ID NO.1, is significantly higher than that of the FLG+ / - group mice. This suggests that the filaggrin mimetic peptide plays a role in improving the water retention of the epidermis of FLG+ / - mice.

[0066] 2. Effects of filaggrin mimetic peptides on mouse epidermis

[0067] HE staining of tissue sections: The mice were anesthetized and killed, and the whole layer of skin tissue was quickly fixed in 4% paraformaldehyde solution. After 24 hours, it was dehydrated with 70%, 80%, 95% and anhydrous ethanol series of alcohol concentrations. After xylene transparent treatment, the tissue sample was immersed in paraffin and embedded in a pre-made embedding frame. The embedded wax block was fixed on the slicer and sliced ​​with a thickness of 5-10 microns. The cut sections were attached to the slide, baked at 60°C for 1 hour, dewaxed and rehydrated with xylene, anhydrous ethanol, 95%, 90%, 80%, and 75% ethanol in turn, and finally washed with distilled water. Then it was stained with hematoxylin-eosin (HE) for 5-10 minutes, washed with distilled water for 1 minute, and differentiated with 0.5% hydrochloric acid ethanol for 10 seconds. Washed with distilled water for 2 minutes, 0.2% ammonia water returned to blue for 40 seconds, washed with distilled water for 1 minute twice, stained with 0.5% eosin for 5 minutes, and washed quickly with distilled water. Finally, seal with neutral gum and observe under a microscope.

[0068] The results are as follows Figure 5 As shown. Figure 5 It can be seen that compared with WT wild-type mice, the epidermis of mice in the FLG+ / -filaggrin deficiency group was thickened, the stratum corneum was slightly thickened, and the reticular structure was destroyed. The epidermis thickness of the mice in the FLG+ / - group coated with the polypeptide SEQ ID NO.1 was significantly reduced compared with the FLG+ / -filaggrin deficiency group.

[0069] ImageJ software was used to measure the epidermal thickness in the HE-stained images. The image scale was set by the scale function, and then the epidermal thickness was traced with a straight line and measured using the "measures" function. The epidermal thickness of 5 different parts of each mouse skin section was measured. GraphPadPrism 10.1.2 software was used to analyze the data and draw a bar graph of the epidermal thickness of each group of mice. The results are shown in the figure. Figure 6 shown.

[0070] Depend on Figure 6 It can be seen that compared with the WT group of mice, the epidermis of the FLG+ / - group of mice was significantly thickened, and the epidermal thickness of the FLG+ / - group coated with the filaggrin mimetic peptide SEQ ID NO.1 was significantly reduced compared with the FLG+ / - group. In dermatitis associated with filaggrin deficiency, epidermal thickening is a typical pathological manifestation. The decrease in epidermal water retention causes keratinocyte proliferation to protect the underlying skin, resulting in epidermal thickening. Frequent scratching can also lead to epidermal cell proliferation and form thickened epidermal tissue. The results of pathological sections show that the application of filaggrin mimetic peptide can significantly alleviate the epidermal thickening caused by filaggrin deficiency.

[0071] 3. Effects of filaggrin mimetic peptides on the stratum corneum of mouse skin

[0072] Gently cut about 0.5×0.5 cm mouse skin, put it into 2.5% glutaraldehyde solution for fixation, then fix it with 1% osmium tetroxide at 4°C, rinse it with 0.1M PBS (phosphate buffer). Then dehydrate it with gradient concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 100%), dry it with a critical point dryer, stick the sample on the sample stage with conductive glue, coat a conductive film on the surface of the sample, and observe the processed sample under a scanning electron microscope. The results are as follows Figure 7 shown.

[0073] Depend on Figure 7 It can be seen that compared with WT wild-type mice, the stratum corneum of the skin of mice in the FLG+ / -filament protein deficiency group was arranged in a disordered manner, the outline of keratinocytes was unclear, and the protrusions and particles increased. This indicates that the lack of filaggrin leads to the destruction of the keratinocyte structure, and the disordered arrangement makes the intercellular space larger, making it easier for water to be lost from the intercellular space of keratinocytes, and it is also easier for bacteria and other foreign substances to invade the deep layer of the epidermis. The epidermal stratum corneum of mice in the FLG+ / - group coated with the polypeptide SEQ ID NO.1 was flatter and more tightly arranged than that in the FLG+ / -filament protein deficiency group, with fewer protrusions and particles, indicating that excessive keratinization was alleviated, and the tight intercellular space was conducive to the recovery of the epidermal water retention function.

[0074] 4. Detection of Filaggrin Mimetic Peptide as Bleomycin Hydrolase (BMH) Substrate

[0075] In order to detect the activity of the filaggrin mimetic peptide as a BMH substrate, the degradation of the filaggrin mimetic peptide was detected in vitro using recombinant human BMH protein (ab101205, Abcam).

[0076] Solid phase synthesis labeled 2 Flag tags (DYKDDDDK) linked to the filaggrin mimetic peptide, labeled as 2Flag-SEQID NO.1, solid phase synthesis labeled 2 Flag tags linked to the non-deiminated filaggrin mimetic peptide, that is, citrulline in SEQ ID NO.1 was replaced with arginine, labeled as 2Flag-R. 100 μg of 2Flag-SEQ ID NO.1 and 2Flag-R were incubated in 50 μL BMH reaction buffer at 37 ° C for 2 h, and the enzymatic reaction was stopped by boiling. The composition of the BMH reaction buffer is: 50 μg recombinant human BMH protein, 50 mM HEPES-NaOH (pH 7.5), 10 mM DTT and 5 mM EDTA.

[0077] The reaction product was added to a 15% SDS-PAGE gel and electrophoresed. After the electrophoresis, the membrane was transferred at 200 mA for 20 minutes, and the protein was transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 hour, and the membrane was incubated with FLAG antibody (M20008, Abmart) at 4°C overnight. After washing the membrane, the HRP secondary antibody was incubated at room temperature for 1 hour, and the chemiluminescence imaging system was used for imaging and the test results were recorded. Figure 8 shown.

[0078] Depend on Figure 8 It can be seen that after 2 hours of incubation, the remaining polypeptides in the 2Flag-R group were significantly more than those in the 2Flag-SEQ ID NO.1 group, indicating that the degradation efficiency of BMH for 2Flag-SEQ ID NO.1 was higher than that of 2Flag-R. The results showed that after replacing the arginine R in the polypeptide GPHQQSHQESARGRSG (SEQ ID NO.15) with citrulline Cit, its efficiency of recognition and degradation by BMH was improved, making it more conducive to being degraded by the natural filaggrin of the epidermis into the metabolic pathway of NMF natural moisturizing factor, and more conducive to the production of natural ingredients that simulate the degradation products of filaggrin.

[0079] Example 4. Application of the filaggrin mimetic peptide of the present invention in skin care cream

[0080] 1. Skin care cream formula (mass ratio): caprylic / capric triglyceride 15%, squalane 5%, lauric acid monoglyceride 3%, glycerol 5%, deionized water 70.2%, allantoin 1%, lactic acid 0.5%, filaggrin mimetic peptide 0.3%.

[0081] 2. Preparation method: Prepare water phase and oil phase solutions separately. Mix water and glycerin to ensure that glycerin is completely dissolved in water, heat to 70°C, add allantoin, stir evenly to obtain water phase.

[0082] In another container, caprylic / caprylic triglyceride, squalane and lauric acid monoglyceride are mixed and heated to about 70° C. until all ingredients are completely melted and mixed uniformly to obtain an oil phase.

[0083] Slowly pour the oil phase into the water phase while stirring continuously until a uniform emulsion is formed. After the mixture cools to room temperature, add 0.3% of the filaggrin mimetic peptide and mix evenly. Finally, add lactic acid and stir evenly, and adjust the pH value to 5-5.5. The filaggrin mimetic peptide and lactic acid are pre-dissolved in a trace amount of deionized water in the formula before mixing, and then mixed with other raw materials.

[0084] Finally, the skin care cream is white and milky, smooth and easy to spread, has good stability, and has no stratification or precipitation.

[0085] 3. Moisturizing effect test

[0086] 0.1 ml of skin cream was used for the experiment, and 10% glycerol solution and deionized water were used as controls. They were applied to the skin on the back of the neck of FLG+ / - mice (n=6). After 4 hours, a non-invasive skin detector (Courage & Khazak, Germany) was used. The CM825 probe detects the water content of the epidermal stratum corneum. The mouse is anesthetized, and the probe is pressed vertically on the treated area of ​​the mouse during the test. The probe is pressed lightly, lifted to make the probe rebound, and the result is read.

[0087] The results are as follows Fig. 9 As shown in the figure, the water content of the stratum corneum of the FLG+ / - mice smeared with the skin cream of the present invention is significantly higher than that of the glycerol solution group and the deionized water control group, indicating that the skin cream has a good moisturizing effect on the skin of FLG+ / - mice lacking filaggrin.

Claims

1. A filaggrin mimetic peptide, characterized in that: The amino acid sequence of the mimetic peptide is SEQ ID NO.

1.

2. Use of the filaggrin mimetic peptide according to claim 1 in a preparation for relieving dry skin.

3. Use of the filaggrin mimetic peptide according to claim 1 in cosmetics.

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