Polypeptide and related application thereof

Abstract

The invention discloses a polypeptide and related application thereof, and relates to the field of biology. The ginseng polypeptide is obtained through screening, the amino acid sequence of the ginseng polypeptide has at least 80% identity with the sequence shown in SEQ ID NO: 1, and the ginseng polypeptide has anti-inflammatory and / or anti-aging effects and the like and can be applied to preparation of products with corresponding effects.

Description

Technical Field

[0001] The present invention relates to the field of biology, and in particular, to a polypeptide and its related applications. Background Art

[0002] Inflammation and aging are two key factors affecting skin health. Chronic inflammation accelerates skin aging, and the loss of collagen (such as COL1A1) during the aging process leads to skin laxity and wrinkle formation. Currently, common anti-inflammatory and anti-aging strategies mainly include: anti-inflammatory drugs (such as glucocorticoids, NSAIDs): Although they can effectively inhibit inflammation, long-term use may cause side effects such as skin atrophy and immunosuppression; antioxidants (such as vitamin C, coenzyme Q10): They can reduce oxidative stress, but have limited direct promotion effects on collagen synthesis; growth factor components (such as TGF-β, EGF): They can promote collagen production, but have problems such as high cost, poor stability, and potential pro-tumor risks.

[0003] In addition, there are few polypeptide molecules in the prior art that can simultaneously have the dual effects of anti-inflammation and promoting collagen regeneration. Most anti-inflammatory components only inhibit inflammatory factors and cannot effectively promote the synthesis of key collagens such as COL1A1; conversely, some collagen-promoting components (such as certain collagen peptides) may exacerbate the inflammatory response due to stimulating the over-activation of fibroblasts. Therefore, developing a new active ingredient that can not only efficiently anti-inflammation but also safely promote collagen regeneration has important market value and clinical significance.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a polypeptide and its related applications.

[0006] The present invention is implemented as follows:

[0007] In the first aspect, an embodiment of the present invention provides a polypeptide, the amino acid sequence of which has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0008] In the second aspect, an embodiment of the present invention provides a biological material, which is selected from an isolated nucleic acid molecule, a vector containing the isolated nucleic acid molecule, or a host cell containing the vector, and the isolated nucleic acid molecule encodes the polypeptide described in the foregoing embodiment.

[0009] In the third aspect, an embodiment of the present invention provides a pharmaceutical composition, which includes: the polypeptide described in the foregoing embodiment.

[0010] Fourthly, the embodiments of the present invention provide the use of the polypeptide as described in the foregoing embodiments, or the biomaterial as described in the foregoing embodiments, or the pharmaceutical composition as described in the foregoing embodiments in the preparation of products for anti-inflammation, anti-aging and / or skin repair.

[0011] The present invention has the following beneficial effects:

[0012] In this application, ginseng polypeptides are screened and obtained. The amino acid sequence thereof has at least 80% identity with the sequence shown in SEQ ID NO: 1, and it has effects such as anti-inflammation and / or anti-aging, and can be applied to the preparation of products with corresponding effects. Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0014] Figure 1 It is the flow chart for screening ginseng peptides;

[0015] Figure 2 It is the detection result of the contents of IL-6 (A), TNF-α (B), and NO (C);

[0016] Figure 3 It is the detection result of the content of Collagen I; among them, A is the content of Collagen I corresponding to 25 μg / mL GPs (Ginseng Peptides) + 15 J UVA, and B is the content of Collagen I corresponding to 15 J UVA;

[0017] Figure 4 It is the detection result of the content of Collagen I. Detailed Embodiments

[0018] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0019] Definition of Terms

[0020] In the case of two or more nucleic acid or polypeptide sequences, the term "identity" percentage refers to the situation where, when compared and aligned for maximum correspondence, two or more sequences or subsequences are identical or have the same amino acid residues or nucleotides at a specified percentage. Sequence alignment methods for comparison are well known in the art. Once aligned, the number of matches is determined by counting the positions where the same nucleotides or amino acid residues are present in the two sequences. The sequence identity percentage is determined by dividing the number of matches by the length of the sequence as stated in the identified sequence, or by the length of the alignment (the length of the sequence region actually participating in the alignment after alignment), and then multiplying the resulting value by 100.

[0021] The best alignment of the sequences for comparison can be carried out by, for example, the following methods: local homology algorithm, Smith and Waterman, Adv. Appl. Math. 2:482, 1981; homology alignment algorithm, Needleman and Wunsch, Mol. Biol. 48:443, 1970; similarity search method, Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI); or manual alignment and visual inspection (see, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley and Sons, New York, supplement 104, 2013)).

[0022] The term "host cell" refers to a cell in which a vector can be propagated and its DNA or RNA can be expressed. The cell can be prokaryotic or eukaryotic.

[0023] The term "treatment" includes preventing or alleviating a condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating the symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0024] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0025] Technical solution

[0026] On the one hand, an embodiment of the present invention provides a polypeptide, the amino acid sequence of which has at least 80% identity with the sequence shown in SEQ ID NO: 1.

[0027] In some embodiments, the at least 80% identity includes any one or the range between any two of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100%.

[0028] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1 (QEGIYPNNDLYRPK), named QK-14. Compared with other ginseng polypeptides, QK-14 has more excellent anti-inflammatory and anti-aging effects.

[0029] On the other hand, an embodiment of the present invention provides a biological material, the biological material is selected from an isolated nucleic acid molecule, a vector containing the isolated nucleic acid molecule, or a host cell containing the vector, and the isolated nucleic acid molecule encodes the polypeptide described in any of the foregoing embodiments.

[0030] In some embodiments, the vector refers to a nucleic acid molecule capable of transporting or transferring an exogenous nucleic acid molecule, including an expression vector and a cloning vector. An "expression vector" refers to a vector capable of expressing an insert in a target cell and usually contains control sequences (such as enhancer, promoter, and terminator sequences) that drive the expression of the insert. The exogenous nucleic acid molecule is called an "insert" or "transgene". A vector usually consists of an insert and a larger sequence serving as the backbone of the vector. Based on the structure or source of the vector, the main types of vectors include plasmid vectors, cosmid vectors, phage vectors (such as λ phage), viral vectors (such as adenovirus (Ad) vectors), and artificial chromosomes.

[0031] The polypeptides provided by the present application can be prepared by conventional methods known in the art, such as expression using a suitable vector in a recombinant host system. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (a clonal isolate derived from the parental Trichoplusia ni BTITN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, which are typically transformed with sheared adenovirus type 5 DNA), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx.RTM. cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as the baculovirus vector system, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for the baculovirus / insect cell expression system are particularly available in kit form from Invitrogen, San Diego, Calif. Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168, and 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known in the art and are described, for example, in Yeast Genetic Engineering (edited by Barr et al., 1989), Butterworths, London.

[0032] On the other hand, embodiments of the present invention provide a pharmaceutical composition, which includes: the polypeptide described in any of the foregoing embodiments.

[0033] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0034] In some embodiments, the pharmaceutically acceptable carrier includes: any one or a combination of more of isotonic saline, ethanol, phosphate buffered saline, balanced salt solution, and dimethyl sulfoxide.

[0035] In addition, the embodiments of the present invention also provide the use of the polypeptide as described in any of the foregoing embodiments, or the biomaterial as described in any of the foregoing embodiments, or the pharmaceutical composition as described in any of the foregoing embodiments in the preparation of products for anti-inflammatory, anti-aging and / or skin repair.

[0036] In some embodiments, the anti-inflammatory effect includes down-regulating the content of any one or more of IL-6, TNF-α and NO.

[0037] In some embodiments, the skin repair and / or anti-aging effect includes promoting the expression of collagen genes.

[0038] In some embodiments, the collagen gene includes the α1 chain of type I collagen.

[0039] In some embodiments, the product includes any one of drugs, cosmetics and skin care products.

[0040] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0041] Example 1. Screening of polypeptides

[0042] 1. Predict the polyproline type of ginseng polypeptides and target proteins through the HSM model

[0043] For the HSM model, specifically refer to Cunningham J M, Koytiger G, Sorger P K, et al. Biophysical prediction of protein–peptide interactions and signaling networks using machine learning[J]. Nature methods, 2020, 17(2): 175-183. The HSM model is a machine learning model that can calculate the possibility of interaction between polypeptides and proteins. The results are given in the form of p value. A value <0.05 is positive, and the lower the value, the higher the possibility of interaction. When using the HSM model for prediction, the polypeptide can be processed in three ways and then three independent predictions can be made: polyproline: directly predict the full length of the peptide; c-terminal: predict the six amino acids at the C-terminal (the rightmost end) of the peptide; phosphosite: take a peptide with a length of 15 and the eighth amino acid being S or T, and replace the S or T at the eighth position with y. For example, for DKSSEVETTDRGLFD, it should be written as DKSSEVEyTDRGLFD and sent into the model for prediction.

[0044] 308 ginseng peptides were obtained (average length of about 12 aa, length range of 5-28 aa), length screening was performed, and peptides with a length of ≤15 aa were selected, and 227 were obtained by filtering.

[0045] There are 38 target proteins (among the targets in the HSM database, those related to the keywords of antioxidant, pro-proliferation, and pro-collagen). The top 50 peptides with the lowest p-values were selected for the polyproline type prediction results. For these 50 peptides, the three lowest proteins were selected according to the p-values, for a total of 150 peptide-target protein pairs.

[0046] 2. Further prediction through CAMP model

[0047] CAMP model, for details, please refer to Lei Y, Li S, Liu Z, et al. A deep-learning framework for multi-level peptide–protein interaction prediction [J]. Nature communications, 2021, 12 (1): 5465. The CAMP model is a deep learning model based on neural networks and attention mechanisms, which can calculate the possibility of interaction between peptides and proteins. The results are given in the form of probabilities within 0-1, > 0.5 is positive, and the higher the value, the higher the possibility of interaction.

[0048] For the 150 peptide-target protein pairs screened in step 1, the CAMP model was used to further predict that 20 pairs were predicted to be positive, involving 11 peptides (screening process see Figure 1 ), see Table 1, which includes QK-14.

[0049] Table 1 Prediction results

[0050]

[0051] Example 2: Anti-inflammatory efficacy experiment of ginseng peptide QK-14

[0052] The inhibitory effect of ginseng peptides on inflammatory factors (IL-6, TNF-α and NO) was detected by ELISA (enzyme-linked immunosorbent assay) to verify its anti-inflammatory efficacy.

[0053] The cell line used in the experiment was RAW264.7.

[0054] Experimental procedures

[0055] (1) Cell culture

[0056] 1.1. Cell Resuscitation: Take out the cell cryopreservation tube, quickly place it in a 37°C water bath and shake until it thaws. Transfer it to a centrifuge tube (containing complete DMEM medium) under a laminar flow hood environment. Then place the centrifuge tube in a centrifuge, balance it, and set the program as follows: rotation speed 800 rpm; time 3 min. After the operation is completed, discard the supernatant, add an appropriate amount of medium (containing serum), gently pipette with a pipette tip, and add it to a T75 culture flask containing 10% serum medium. Mix well and place it in a cell culture incubator for static culture.

[0057] 1.2. Cell Passage: Two days after cell resuscitation, take out the T75 culture flask, observe it under a microscope. When the cell density is about 80%-90% (do not let it reach confluence), discard the supernatant in the laminar flow hood, wash it once with PBS, add 2 ml of trypsin, incubate in the incubator for 20 s, then add 2 ml of complete DMEM medium to terminate the digestion. Pipette the cells at the bottom of the culture dish, collect them into a centrifuge tube, and centrifuge them using the same centrifuge (with the same program as before). After centrifugation, discard the supernatant, add 2 ml of complete medium to resuspend the cells, pipette evenly with a pipette tip, and then aspirate 400 μl and add it to a T75 culture flask containing 10 ml of complete DMEM medium. Mix well and place it in a cell culture incubator for static culture.

[0058] 1.3. Cell Cryopreservation: Take out the T75 culture flask, discard the medium, wash it once with PBS, add 2 ml of trypsin, incubate in the incubator for 20 s, then add 2 ml of complete DMEM medium to terminate the digestion. Pipette the cells at the bottom of the culture dish, collect them into a centrifuge tube, and centrifuge them using the same centrifuge (with the same program as before). After centrifugation, discard the supernatant, add 4 - 5 ml of cell cryopreservation solution (serum:DMSO:medium = 2:1:7) to resuspend the cells, and then add 1 ml of cell cryopreservation solution to each cryopreservation tube. Place the cryopreservation tubes in a programmable cooling box and transfer them to an -80°C refrigerator for storage.

[0059] (2) Group Treatment

[0060] 2.1. Plating: Take out the T75 culture flask, discard the medium, wash it once with PBS, add 2 ml of trypsin, incubate in the incubator for 20 s, then add 2 ml of complete DMEM medium to terminate the digestion. Pipette the cells at the bottom of the culture dish, collect them into a centrifuge tube, and centrifuge them using the same centrifuge (with the same program as before). After centrifugation, discard the supernatant, add 2 ml of complete DMEM medium, resuspend the cells, then take out 180 μl, add 20 μl of trypan blue staining solution, and use a countstar cell counter to count the number of cells. Finally, add the complete medium containing the cells to a 24-well plate, and the final cell density is 5×10 4 cells / well. After plating, place it in a cell culture incubator and culture for 24 h.

[0061] 2.2. Induced sample addition: Induced model construction: When the cell confluence reaches about 70%, discard the culture medium, add DMEM complete medium containing LPS, and simultaneously add a certain concentration of the test substance. The grouping and treatment are as follows, and incubate for 24 h.

[0062] Blank control group (control): Complete medium;

[0063] Inflammatory model group (Model): Stimulated with LPS (200 ng / mL);

[0064] Ginseng peptide drug addition group: LPS (200 ng / mL) + complete medium containing different concentrations of ginseng peptide (10, 25 μg / mL).

[0065] Positive control group: LPS (200 ng / mL) + complete medium containing 50 μg / mL dexamethasone.

[0066] (3) Sample collection

[0067] After culturing in the incubator for 24 h, collect the cell culture medium into a 1.5 mL centrifuge tube and store it in a -80 °C refrigerator.

[0068] (4) ELISA experiment

[0069] According to the detection instructions of IL-6, TNF-α, and NO, detect the concentrations of IL-6, TNF-α, and NO. Set three replicates for each sample.

[0070] The results are shown in Figure 2 , compared with the blank group, after treatment with 200 ng / mL LPS for 24 h, the contents of IL-6, TNF-α, and NO in the model group increased significantly. After treatment with Dex (dexamethasone), the contents decreased, and the model was successfully established.

[0071] After treatment with 10, 25 μg / mL QK-14, the contents of IL-6, TNF-α, and NO in the cell culture supernatant decreased with the increase of the polypeptide concentration, and showed a concentration dependence. After treatment with 25 μg / mL QK-14, IL-6, TNF-α, and NO decreased by 14.7%, 23.4%, and 29.3% respectively.

[0072] Example 3. Anti-aging efficacy experiment of ginseng peptide QK-14

[0073] Detect the promoting effect of ginseng peptide on COL1A1 (alpha-1 chain of type I collagen) by ELISA method to verify its anti-skin aging efficacy.

[0074] Cell line: HSF.

[0075] Experimental procedure (1) Cell culture and treatment

[0076] 1.1. Cell culture

[0077] (1) Cell resuscitation: Take out the cell cryopreservation tube, quickly place it in a 37°C water bath and shake until it melts. Transfer it to a centrifuge tube (containing complete DMEM medium) under a laminar flow hood environment. Then place the centrifuge tube in a centrifuge, balance it, and set the program as follows: rotation speed 800 rpm; time 3 min. After the operation is completed, discard the supernatant, add an appropriate amount of medium (containing serum), gently pipette with a pipette tip, add it to a 10 ml T75 culture flask containing complete medium, mix well and place it in a cell culture incubator for static culture.

[0078] 1.2. Cell passage: Two days after cell resuscitation, take out the T75 culture flask, observe it under a microscope. When the cell density is about 90%, discard the supernatant in the laminar flow hood, wash it once with PBS, add 3 ml of trypsin, let it stand and digest in the incubator for 60 s, then add 3 ml of complete DMEM medium to terminate the digestion. Pipette the cells at the bottom of the culture dish, collect them into a centrifuge tube, and centrifuge them in the same centrifuge (with the same program as before). After centrifugation, discard the supernatant, add 2 ml of complete medium to resuspend the cells, pipette them evenly with a pipette tip and aspirate 1 ml into a T75 culture flask containing 10 ml of complete DMEM medium, mix well and place it in a cell culture incubator for static culture.

[0079] 1.3. Cell cryopreservation: Take out the T75 culture flask, discard the medium, wash it once with PBS, add 3 ml of trypsin, let it stand and digest in the incubator for 60 s, then add 3 ml of complete DMEM medium to terminate the digestion. Pipette the cells at the bottom of the culture dish, collect them into a centrifuge tube, and centrifuge them in the same centrifuge (with the same program as before). After centrifugation, discard the supernatant, add 2 ml of cell cryopreservation solution (serum:DMSO:medium = 2:1:7) to resuspend the cells, and then add 1 ml of cell cryopreservation solution to each cryopreservation tube. Put the cryopreservation tube into a programmable cooling box and transfer it to an -80°C refrigerator for storage.

[0080] (2) Group treatment

[0081] 2.1. Plating: Take out the T75 culture flask, discard the medium, wash it once with PBS, add 3 ml of trypsin, let it stand and digest in the incubator for 60 s, then add 3 ml of complete DMEM medium to terminate the digestion. Pipette the cells at the bottom of the culture dish, collect them into a centrifuge tube, and centrifuge them in the same centrifuge (with the same program as before). After centrifugation, discard the supernatant, add 2 ml of complete DMEM medium, resuspend the cells and take out 180 μl, add 20 μl of trypan blue staining solution, use a countstar cell counter to count the number of cells, and finally add the medium containing the cells to a 12-well plate. The final cell density is 4×10 4 cells / well. After plating, place it in a cell culture incubator for 24 h.

[0082] 2.2. Sample addition:

[0083] a. Normal sample addition: When the cell confluence reaches about 70%, discard the culture medium, add DMEM complete glucose medium containing a certain concentration of the test substance or positive control, and set up groups as shown below. Incubate for 24 h.

[0084] b. UV-induced sample addition: When the cell confluence reaches about 70%, discard the culture medium, add PBS containing a certain concentration of the test substance or positive control, irradiate with ultraviolet light (15 J / cm 2 , and the irradiation intensity should not be too high), and then replace it with DMEM complete medium containing a certain concentration of the test substance or positive control.

[0085] Blank control group (Control): Complete medium (without irradiation);

[0086] Aging model group (Model): Irradiation + complete medium;

[0087] Ginseng peptide drug addition group: Irradiation + complete medium containing different concentrations of ginseng peptide (10, 25 μg / mL);

[0088] Positive control group: Irradiation + complete medium containing 100 ng / mL TGF-β1.

[0089] (3) Sample collection

[0090] After culturing in the incubator for 24 h, collect the cell culture and store it in a 1.5 mL centrifuge tube at -80 °C.

[0091] (4) Detection of type I collagen content

[0092] According to the ELISA detection instructions for type I collagen, detect the concentration of type I collagen, and set three replicates for each sample.

[0093] The results are shown in Figure 3 .

[0094] Compared with the blank group, after UVA irradiation, the content of COL1A1 in the model group decreased significantly.

[0095] After treatment with 10, 25 μg / mL QK-14, the content of COL1A1 in the cell culture supernatant increased with the increase of the polypeptide concentration, showing a concentration dependence. After treatment with 25 μg / mL QK-14, the content increased by 268.1%.

[0096] Example 4. Anti-aging efficacy experiment of ginseng peptide QK-14

[0097] Detect the promoting effect of ginseng peptide on COL1A1 (alpha 1 chain of type I collagen) by ELISA method to verify its anti-skin aging efficacy.

[0098] Cell lines: THP-1 & HSF.

[0099] Experimental procedures

[0100] (1) Cell culture

[0101] THP-1 cells are suspension cells and can be resuscitated with RPMI 1640 complete medium and passaged by semi-changing or full-changing the medium. HSF cells are adherent cells and can be resuscitated and passaged with DMEM complete medium. THP-1 cells and HSF cells are cultured in cell culture dishes or flasks in a cell culture incubator at 37°C and 5% CO2.

[0102] When using cryopreserved cells, follow the principle of slow freezing and rapid thawing. Passage the cells at least 2 times after resuscitation before use. Cells from the same source and the same passage number should be selected for the experiment to ensure the consistency of experimental results.

[0103] (2) Preparation

[0104] 2.1 Preparation of test substances

[0105] Test substances with good water solubility can be directly dissolved in ddH2O to prepare a high-concentration stock solution, and then diluted to the working concentration with complete medium. Test substances with limited solubility in water should be dissolved in an appropriate solvent. The volume of the solvent should be consistent in all cultures, that is, consistent in the blank control and the test substance, and non-cytotoxic. Test substances with limited water solubility are preferably dissolved in dimethyl sulfoxide (DMSO) as the solvent. However, during cell culture, the final concentration of dimethyl sulfoxide (DMSO) should generally be less than 0.1% - 1% (v / v), and should be adjusted according to the results of cytotoxicity experiments; vortex mixing, sonication or heating can be used but are not limited to assist in dissolution to prepare a solution or a homogeneous suspension; test substances that cannot be prepared into a solution or a homogeneous suspension are not applicable to this test. When selecting a solvent, their special properties should be considered, but not limited to whether they react with the test substance and their chemical stability in the solution. When selecting the concentration of the test substance, precipitation or turbidity of the solution should be avoided. Complete medium (containing additives) should not be used to prepare the stock solution to avoid precipitation caused by repeated freezing and thawing of serum proteins, test substances or other components.

[0106] 2.2 Preparation of cell culture medium

[0107] RPMI 1640 complete medium: RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin double antibody, and 0.05 mM β-mercaptoethanol.

[0108] DMEM complete medium: DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin double antibody.

[0109] (3) Test conditions

[0110] 3.1, Concentration of the test substance

[0111] The concentration range of the test substance should be determined by cytotoxicity detection. Select the concentration at which the cell survival rate reaches about 90% as the starting concentration, and set 1 to 3 concentrations below it as the concentrations for the formal test. The specific concentration selection can be appropriately adjusted according to the test situation. The final concentration of the solvent should be kept consistent, without cytotoxicity, and should not have any other effects on cell proliferation and differentiation.

[0112] 3.2, Positive control

[0113] TGF-β1 is used as the positive control group in each test. The content of type I collagen is measured by the in vitro macrophage fibroblast model test method. The recommended test concentration of TGF-β1 is 100 ng / mL, which is diluted 1000 times from a stock solution with a concentration of 100 μg / mL.

[0114] (4) Test procedures

[0115] 4.1, Induction of THP-1 cells

[0116] Select THP-1 cells that are in good growth and within 10 passages of the logarithmic growth phase, and inoculate them into a 6-well plate at a density of 1×10 6 / well (2 mL of complete cell culture medium per well), or select a cell culture plate of appropriate specifications according to the experimental conditions. Treat for 24 h to induce the formation of pseudopodia and adherence to obtain M0 macrophages. Remove the culture medium, wash each well twice with 1 mL of PBS, add 3 mL of RPMI 1640 complete culture medium to each well, and culture for 2 d to collect the culture supernatant of M0 macrophages.

[0117] Prepare RPMI 1640 complete culture medium containing 100 nM PMA, 100 ng / mL LPS, and 20 ng / mL IFN-γ (prepared immediately before use). Remove the original culture medium of M0 macrophages, add 1 mL of the above complete culture medium to each well, and continue to treat for 24 h to obtain M1 macrophages. Remove the culture medium, wash each well twice with 1 mL of PBS, add 3 mL of RPMI 1640 complete culture medium to each well, and culture for 2 d to collect the culture supernatant of M1 macrophages.

[0118] 4.2, Collection and preservation of culture supernatants of M0 and M1 macrophages

[0119] Collect the culture supernatant of M0 or M1 macrophages at the corresponding time points in step 4.1, store it in a -80°C refrigerator, and perform subsequent tests within one week.

[0120] 4.3, HSF Cell Culture and Seeding

[0121] Select HSF cells that are in good growth condition and within 10 generations of the logarithmic growth phase, and seed them into a 12-well plate at a density of 5×10 4 cells / well (1 mL of complete medium containing cells per well), or select a cell culture plate of appropriate specifications according to experimental conditions. Set 3 replicates for each group. After seeding, place it in a CO2 incubator and culture for 24±2 h.

[0122] 4.4, HSF Cell Induction

[0123] For each batch of experiments, set a test substance group, a blank control group (Control), a model group (Model), and a positive control group, with 3 replicates for each group. Discard the culture medium, add the culture supernatant of M1 macrophages containing the test substance (10, 25 μg / mL QK-14) to the test substance group, add the culture supernatant of M1 macrophages to the model group, add the culture supernatant of M1 macrophages containing 100 ng / mL TGF-β1 positive control to the positive control group, and add normal RPMI1640 complete medium to the blank control group, 1 mL per well. After adding the samples, place the cell culture plate in a CO2 incubator and culture for 3 d (when the cell confluence is below 95%), and collect the culture supernatant for testing.

[0124] 4.5, Determination of Type I Collagen Content in HSF Cells

[0125] According to the operation instructions of the human type I collagen ELISA detection kit, determine the type I collagen content in the culture supernatant of each group of HSF cells.

[0126] The results are shown in Figure 4 .

[0127] Compared with the blank group, the content of COL1A1 in the model group was significantly decreased. After treatment with 10, 25 μg / mL QK-14, the content of COL1A1 in the cell culture supernatant increased with the increase of polypeptide concentration and showed concentration dependence. The up-regulation rate was 35.9% after treatment with 10 μg / mL QK-14, and 51.0% after treatment with 25 μg / mL QK-14.

[0128] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence has at least 80% identity with the sequence shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, wherein The amino acid sequence of the polypeptide is as shown in SEQ ID NO:

1.

3. A biological material, the biological material being selected from an isolated nucleic acid molecule, a vector containing the isolated nucleic acid molecule, or a host cell containing the vector, characterized in that, The isolated nucleic acid molecule encodes the polypeptide according to claim 1 or 2.

4. A pharmaceutical composition, characterized in that, It includes: The polypeptide according to claim 1 or 2.

5. The pharmaceutical composition according to claim 4, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier; Optionally, the pharmaceutically acceptable carrier includes any one or a combination of more of: isotonic saline, ethanol, phosphate buffered saline, balanced salt solution, and dimethyl sulfoxide.

6. Use of the polypeptide according to claim 1 or 2, or the biological material according to claim 3, or the pharmaceutical composition according to claim 4 or 5 in the preparation of a product for anti - inflammation, anti - aging and / or skin repair.

7. The application according to claim 6, characterized in that, The anti - inflammation includes down - regulating the content of any one or more of IL - 6, TNF - α and NO.

8. The application according to claim 6, wherein The skin repair and / or anti - aging includes promoting the expression of collagen genes.

9. The application according to claim 8, wherein The collagen genes include the α1 chain of type I collagen.

10. The application according to claim 6, characterized in that, The product includes any one of: drugs, cosmetics and skin care products.

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