A humanized collagen type xvn polypeptide, and preparation method and application thereof

Abstract

The application belongs to the technical field of protein engineering, and discloses a humanized collagen type ⅩⅦ polypeptide and a preparation method and application thereof. The amino acid sequence of the humanized collagen type ⅩⅦ polypeptide is shown as SEQ ID NO. 1, and the polynucleotide sequence encoding the humanized collagen type ⅩⅦ polypeptide is shown as SEQ ID NO. 2. The application also provides application of the polypeptide in preparation of a protein product with the functions of preventing hair loss, repairing, anti-wrinkle and skin tightening. The efficacy detection at the cell level proves that the prepared humanized collagen type ⅩⅦ polypeptide can promote the proliferation of hair papilla cells, keratinocytes and fibroblasts, and promote the up-regulation of the expression of the β-Catenin signal pathway protein closely related to hair growth in the hair papilla cells, thereby proving that the humanized collagen type ⅩⅦ polypeptide has the efficacy of preventing hair loss and / or repairing, anti-wrinkle and skin tightening.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of protein engineering, and particularly relates to a humanized collagen type ⅩⅦ polypeptide and a preparation method and application thereof. BACKGROUND

[0002] Collagen is a vital structural protein, and is one of the most abundant and widely distributed proteins in mammals and many organisms, which plays a core role in maintaining the structural support, stability and elasticity of tissues. Collagen widely exists in tissues such as bones, joints, muscles, skin, blood vessels and corneas, and is the main constituent of these tissues. Collagen forms molecular cables with its unique triple helix structure to strengthen tendons and support the membranes of skin and internal organs. In addition to structural support, collagen can also act as a signal molecule to regulate cell proliferation, differentiation and migration, which is crucial for maintaining normal physiological functions and overall health of the body. Recombinant collagen has a wide range of applications in the biomedicine, cosmetics, food and other industries due to its low toxicity, low antigenicity, low immunogenicity, ability to guide cell regeneration and good biocompatibility. In the field of medical beauty, collagen is also an important component for improving skin condition and maintaining skin youth and elasticity.

[0003] Type ⅩⅦ collagen has a molecular weight of 180 kDa, is a transmembrane protein located in the epidermal basement membrane region, is encoded by COL17A1 gene, and has its C-terminal in the extracellular matrix and its N-terminal in the cytoplasm. It is mainly composed of intracellular region, transmembrane structure region and extracellular region. The transmembrane structure region in type ⅩⅦ collagen structure has a non-helical connecting domain (NC16A) with many protease hydrolysis sites, and this region can be hydrolyzed and detached from the cell surface under the action of protease. Due to such structural characteristics, type ⅩⅦ collagen can exist in the form of transmembrane or detachment in the body, and thus has the dual functions of cell membrane surface receptor and extracellular matrix component. In addition, type ⅩⅦ collagen is also a component of hemidesmosomes in cells, and plays an important role in the action of epithelial cells-basement membrane, including regulating the adhesion, separation and developmental differentiation of epithelial cells, and maintaining the homeostasis of related stem cells in skin and hair growth.

[0004] Since it is difficult to extract collagen XVII from animals or human bodies, all the collagen XVII reported in the patents and literatures is obtained by recombinant expression. The main methods for recombinant expression of collagen XVII are intracellular expression in Escherichia coli or secretory expression in Pichia pastoris. The former has problems such as high residual amount of endotoxin, nucleic acid, host protein and other substances in the finished product, complicated protein purification steps, and high cost; and the latter has problems such as low expression amount and easy degradation of the target protein. Moreover, the molecular weight of the collagen XVII reported in most patents and literatures is more than 30 kDa, and the collagen XVII also has the problem of not easily penetrating the skin barrier to reach the dermis to play a role.

[0005] Therefore, it is urgent to find a collagen XVII with small molecular weight and capable of easily penetrating the skin barrier, so as to improve the utilization rate of collagen XVII. SUMMARY

[0006] In order to overcome the above technical problems, the amino acid sequence of collagen XVII is analyzed and optimized in the present application, and a high-activity functional core region in the triple helix structure of collagen is successfully identified and screened, thereby providing a humanized collagen XVII polypeptide with a molecular weight of 2.9 kDa.

[0007] In a first aspect, the present application provides a humanized collagen XVII polypeptide, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

[0008] In a second aspect, the present application further provides a polynucleotide encoding the polypeptide of the first aspect, wherein the sequence of the polynucleotide is shown as SEQ ID NO. 2.

[0009] In a third aspect, the present application further provides an expression vector comprising the polynucleotide of the second aspect.

[0010] In a fourth aspect, the present application further provides a host cell comprising the expression vector of the third aspect.

[0011] Further, the host cell is an Escherichia coli cell.

[0012] In a fifth aspect, the present application further provides a preparation method of the polypeptide of the first aspect, wherein the preparation method comprises,

[0013] 1) culturing the host cell of the fourth aspect in a production medium; and

[0014] 2) isolating the polypeptide from the host cell.

[0015] In a sixth aspect, the present application further provides a composition having the function of preventing hair loss and / or having the function of repairing, anti-wrinkle and skin tightening, wherein the composition comprises the polypeptide according to the first aspect of the present application.

[0016] In a seventh aspect, the present application further provides a protein product having the function of preventing hair loss and / or having the function of repairing, anti-wrinkle and skin tightening, wherein the protein product comprises the polypeptide according to the first aspect of the present application or the composition according to the sixth aspect of the present application, and the protein product is a pharmaceutical composition, a medical device, a tissue engineering product or a cosmetic.

[0017] Further, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0018] In the present application, the "pharmaceutically acceptable carrier" is well known to those skilled in the art, and those skilled in the art can select a pharmaceutically acceptable carrier suitable for the composition or article of the present application. For example, the pharmaceutically acceptable carrier includes but is not limited to: buffers such as phosphoric acid, citric acid and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0019] In an eighth aspect, the present application further provides use of the polypeptide according to the first aspect of the present application, or the polynucleotide according to the second aspect of the present application, or the expression vector according to the third aspect of the present application, or the host cell according to the fourth aspect of the present application, or the composition according to the fifth aspect of the present application in the preparation of a protein product having the function of preventing hair loss and / or having the function of repairing, anti-wrinkle and skin tightening, wherein the protein product is a pharmaceutical composition, a medical device, a tissue engineering product or a cosmetic.

[0020] In the present application, the medical device refers to an instrument, equipment, appliance, in vitro diagnostic reagent and calibrator, material and other similar or related articles which are used directly or indirectly for the human body.

[0021] The present application has the following technical effects relative to the prior art.

[0022] 1) The application successfully identifies and screens a high-activity functional core region in the triple helix structure of collagen type VII from natural collagen type VII through bioinformatics, humanized collagen type VII polypeptide A, the molecular weight of the functional core region is only 2.9 kDa, which is more conducive to transdermal absorption. In addition, the 100% humanized amino acid sequence design of the polypeptide ensures the biocompatibility of the product with human skin and maximally reduces the risk of allergic reactions.

[0023] 2) The prepared humanized collagen type VII polypeptide can promote the proliferation of hair papilla cells, keratinocytes and fibroblasts and promote the up-regulation of the expression of related proteins in the β-Catenin signaling pathway closely related to hair growth in the hair papilla cells through efficacy detection at the cellular level, thereby proving that the humanized collagen type VII polypeptide has the effects of preventing hair loss, promoting hair growth and / or repairing, anti-wrinkle and skin tightening.

[0024] 3) The present application selects an E. coli secretion expression system for the construction and expression of recombinant humanized collagen type VII, compared with traditional E. coli intracellular expression and Pichia pastoris secretion expression, the E. coli secretion expression system transports the recombinant protein to the periplasm, reduces the pollution of host proteins, endotoxins and nucleic acids and the like, simplifies the downstream purification, and is also conducive to improving the stability and expression amount of the target protein. BRIEF DESCRIPTION OF DRAWINGS

[0025] The accompanying drawings, which are included to provide a further understanding of the application and constitute a part of this application, do not limit the application in any way.

[0026] Figure 1 Figure 1 is the analysis of the hydrophilicity and surface charge of humanized collagen type VII polypeptide A; wherein A is a surface charge distribution diagram, and B is a hydrophilicity and hydrophobicity analysis.

[0027] Figure 2 Figure 2 is the expression of humanized collagen type VII polypeptide A in BL21 (DE3); wherein M: protein marker; lane 1: shake flask fermentation supernatant; lane 2: bacterial extraction supernatant.

[0028] Figure 3 Figure 3 is the purification result of humanized collagen type VII polypeptide A; wherein M: protein marker; lane 1: sample prepared by purification of humanized collagen type VII polypeptide A.

[0029] Figure 4 Figure 4 is the experimental result of humanized collagen type VII polypeptide A promoting the proliferation of hair papilla cells.

[0030] Figure 5 Figure 5 is the experimental result of humanized collagen type VII polypeptide A promoting the proliferation of keratinocytes.

[0031] Figure 6 Results of the experiment of humanized collagen type XI polypeptide A promoting fibroblast proliferation.

[0032] Figure 7 Effects of humanized collagen type XI polypeptide A on transcriptional level expression changes of hair papilla cells. DETAILED DESCRIPTION

[0033] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods not specified in the following examples are generally carried out according to the conventional conditions, the conditions described in the laboratory manual or the conditions suggested by the manufacturer.

[0034] The following will be described in detail in conjunction with examples.

[0035] Example 1 Sequence design of humanized collagen type XI polypeptide A

[0036] The amino acid sequence of natural human type XI collagen alpha 1 chain (GenBank: KAI4077360.1) was obtained through the NCBI database, and systematic bioinformatics deep analysis was carried out. Based on the results of multidimensional analysis, the natural human type XI collagen alpha 1 chain was redesigned to obtain a recombinant humanized type XI collagen with higher expression efficiency and structural stability while maintaining high biological activity, which was named polypeptide A, and its amino acid sequence is shown as SEQ ID NO. 1. As shown in the table, the surface charge distribution and hydrophilicity analysis results show that the protein has excellent water solubility and stability. At the same time, humanized collagen type XI polypeptide A contains the biological activity site of natural human type XI collagen alpha 1 chain, and does not contain any tag and other exogenous amino acids, which is a recombinant humanized collagen protein, ensuring the realization of the biological function of the recombinant collagen protein, and has the characteristics of high biocompatibility and low use risk. Figure 1 Example 2 Construction of humanized collagen type XI polypeptide A expression strain

[0037]

[0038] ​According to the codon bias of E. coli, the amino acid sequence described in SEQ ID NO. 1 in Example 1 was reverse translated into a DNA sequence, the nucleotide sequence of which is shown in SEQ ID NO. 2, and a signal peptide SP1 was added at the N-terminus, the amino acid sequence of which is shown in SEQ ID NO. 3, the nucleotide sequence of which is shown in SEQ ID NO. 4, and the sequence was named SP1-A. The DNA sequence was synthesized by Jiangsu Saixuefei Biological Technology Co., Ltd., and cloned into the vector pET-29a, i.e. the recombinant plasmid pET29a-SP1-A. The recombinant plasmid was transformed into E. coli BL21 (DE3), and cultured on a LB solid plate containing kan resistance at 37°C overnight.

[0039] Example 3 Induction expression of recombinant bacteria

[0040] A single colony of recombinant E. coli BL21 (DE3) / pET29a-SP1-A was picked from the transformation plate and inoculated in 5 mL of liquid LB containing a final concentration of 50 μg / mL of antibiotic kan, and cultured at 37°C with 220 rpm shaking for about 5 h. Then, 1% of the inoculum was transferred to 300 mL of liquid LB containing a final concentration of 50 μg / mL of antibiotic kan, and cultured at 37°C with 220 rpm shaking until OD600=0.8. Then, 0.2 mM of IPTG was added to the culture to induce protein expression, and the culture was incubated at 30°C with 220 rpm shaking for about 16 h.

[0041] Cell disruption: 1) 4 mL of bacterial solution was collected in a 2 mL centrifuge tube, centrifuged at 12000 rpm for 1 min, and the bacterial body was taken. 2) After discarding the supernatant, 1 mL of PBS buffer was added to resuspend the cells, which were then broken by ultrasonic waves, and then centrifuged at 12000 rpm for 10 min. 3) After centrifugation, 200 μl of supernatant was taken, and 40 μl of loading buffer was added; the precipitate was resuspended with 400 μl of PBS buffer, and 80 μl of loading buffer was added, mixed well, then boiled in a water bath for 5 min, and then centrifuged at 12000 rpm for 5 min.

[0042] Extraction treatment: 1) 25 mL of bacterial solution was collected and centrifuged at 10000 rpm for 3 min, and the bacterial body was taken. 2) After resuspending the bacterial body with 3 mL of extraction buffer (5 mM EDTA, 100 mM Tris-HCl, pH 8.0), the mixture was shaken at 220 r / min at 37°C for 90 min, and then centrifuged at 12000 rpm for 10 min. 3) After centrifugation, 200 μl of supernatant was taken, and 40 μl of loading buffer was added, mixed well, then boiled in a water bath for 5 min, and then centrifuged at 12000 r / min for 5 min.

[0043] The results are shown in Table 1. Figure 2As shown, due to the presence of some post-expression protein modifications, such as phosphorylation, glycosylation, post-translational cleavage or protein dimerization, etc., the electrophoretic results may have errors. After further verification by molecular biology experiments, it is proved that the polypeptide A is successfully expressed in E. coli and has a high expression level.

[0044] Example 4 Purification and preparation of humanized collagen type XlV polypeptide A

[0045] The fermentation broth in Example 3 was collected and extracted, and the supernatant was collected by centrifugation at 12000 rpm for 10 min. The pH was adjusted to 4.0 using dilute hydrochloric acid, and a cation exchange column SP FF was used with a detection wavelength of 280 nm and 220 nm and a flow rate of 5 ml / min. The column was equilibrated with mobile phase A (20 mM NaAc, pH 4.0), and when A280 nm and the conductance value were stable, the sample was ready for loading. The sample loading flow rate was set to 5 ml / min, and the ultraviolet A280 was detected. When it rose, the sample was loaded, i.e. flow-through sample. After the loading was completed, the column was washed with 5 CV of mobile phase A, and the ultraviolet curve was washed to a stable baseline. Finally, the target protein was eluted using mobile phase B (20 mM NaAc, 1000 mM NaCl, pH 4.0). The eluate was collected and prepared for SDS-PAGE electrophoresis. The results are shown in Figure 3 As shown, the purity of the purified and prepared humanized collagen type XlV polypeptide A sample reached the electrophoretic purity level.

[0046] Example 5 Cell proliferation experiment

[0047] Experimental procedure: Take the cells (HFDPCs, HaCat, HDF) in the logarithmic growth phase (cell density is 80%-90%) and culture them in DMEM / high glucose DMEM complete medium (10% high-quality fetal bovine serum, 1% double-antibiotic), and then inoculate them in a 96-well plate at a density of 5*10 3 cells / well. Add 100 μL of complete medium to each well. After incubation in a constant-temperature incubator at 37 ℃, 5% CO2 and 95% humidity for 24 h (cell density is 70%-80%), add the detection sample (n=5) diluted with complete medium to each well, respectively. The control group is added with serum-free DMEM medium. After incubation for 48 h, discard the culture medium, wash twice with PBS, dilute CCK8 according to the ratio of 100 μL base medium per well + 10 μL CCK8 (avoid light), and slowly add 110 μL of the diluted solution to each well to avoid the formation of bubbles to affect the reading of the enzyme marker. Continue to incubate for 1.5 h (optimal OD is 0.8-1.0), and then read the absorbance value at 450 nm wavelength.

[0048] Data processing and experimental results: use statistical software (Excel, Graphpad, etc.) to calculate the average and standard error of each drug concentration under the multi-well, according to the formula CVR= (A-A0) / (B-A0) x 100% to calculate the cell viability (CVR, %, cell viability ratio) under different drug concentrations (A: treatment group absorbance; B: control group absorbance A0: blank group absorbance).

[0049] Results as shown in Figure 4 , humanized collagen type ⅩⅦ polypeptide A has better proliferative effect on hair follicle cells (HFDPCs), and is better than the effect of competitor 1 (Recol 17 ® , purchased from Jiangsu Chuangjian Medical Technology Co., Ltd.), the results show that humanized collagen type ⅩⅦ polypeptide A has good anti-hair loss effect.

[0050] Results as shown in Figure 5 , Figure 6 , humanized collagen type ⅩⅦ polypeptide A has better proliferative effect on keratinocytes (HaCat), slightly better than the effect of competitor 1 (Recol 17 ® ), the polypeptide has better proliferative effect on fibroblasts (HDF), the results show that humanized collagen type ⅩⅦ polypeptide A has the effect of repairing, anti-wrinkle and tightening skin.

[0051] Example 6 Effect of humanized collagen type ⅩⅦ polypeptide A on transcription level of different cells

[0052] β-Catenin is a multifunctional protein encoded by the gene CTNNB1, which is a key transcription factor in the Wnt / β-catenin signaling pathway. This signaling pathway is the main switch to regulate hair growth. Wnt-mediated hair regeneration involves the stabilization of low-phosphorylated β-catenin, which then binds to TCF / LEF to enter the nucleus and activate target genes to promote cell proliferation. Therefore, this example evaluates the function of humanized collagen type ⅩⅦ polypeptide A by detecting the expression level of related proteins in the Wnt / β-catenin signaling pathway.

[0053] Experimental steps

[0054] Preparation of samples: take the cells in logarithmic growth phase at 6*10 5The cells were inoculated in 6-well plates and cultured at 37°C, 5% CO2 and 95% humidity in a constant temperature incubator for 24 h (confluence at 80-90%), then the culture medium was discarded, washed twice with PBS, and the residual culture medium was washed away, 2 mL of the detection sample diluted with serum-free medium to a final concentration of the detection concentration was added to each well, the detection concentration was 100 ppm in this experiment, and serum-free medium was used as a control group. It was placed in a 37°C, 5% CO2 incubator, and sampled 24 h after administration.

[0055] Cell pretreatment: each experimental sample well was washed twice with PBS, and the liquid was aspirated clean, then 0.5 ml of Trizol was added to each well, and the cells were blown and aspirated repeatedly to blow off the adherent cells, and the sample was transferred to an RNase-free 1.5 ml centrifuge tube.

[0056] Extraction of RNA:

[0057] 1) After taking the sample tube out of the refrigerator, lyse for 5 min at room temperature, shake up and down to lyse thoroughly, do not use vortex to shake, so as to avoid breaking the genomic RNA; centrifuge at 12000 rpm for 5 min at 4°C, and transfer the supernatant to a new RNase-free 1 ml EP tube.

[0058] 2) Add 100 μl of 4°C pre-cooled chloroform (trichloromethane) to each EP tube (0.2 ml is added to 1 ml Trizol), shake vigorously for 15 s, then incubate at room temperature for 5 min, centrifuge at 12000 rpm for 15 min at 4°C, and take the upper 60% water sample. Use a 200 μl syringe to take about 400-500 μl. After adding chloroform, there are three layers: water phase, middle white layer, and lower organic phase.

[0059] 3) RNA elution: add 1 μl of GLycogen to each tube, then add 250 μl of 4°C pre-cooled isopropanol, mix gently, and incubate at room temperature for 5-10 min.

[0060] 4) Centrifuge at 12000 rpm for 10 min at 4°C, and slowly aspirate the supernatant with a pipette. The RNA is present at the bottom of the tube, and try to aspirate it clean.

[0061] 5) RNA washing: add 0.5 ml of 75% pre-cooled ethanol to each EP tube (same amount as Trizol), gently invert the centrifuge tube wall to suspend the precipitate.

[0062] 6) Centrifuge at 8000 rpm for 5 min at 4°C, and slowly aspirate the supernatant with a pipette.

[0063] 7) Add 1 ml of 75% ethanol for washing.

[0064] 8) Centrifuge at 8000 rpm for 5 min at 4°C, and slowly aspirate the supernatant with a pipette.

[0065] 9)Invert the centrifuge tube and let the ethanol evaporate for 10 min.

[0066] 10)Add 40 μl DEPC water to each EP tube to dissolve the sample, and vortex for 10 s.

[0067] 11)Determine the A260 / A280 and RNA concentration of each sample, and store at -20℃ for later use.

[0068] Reverse transcription of cDNA, according to the reverse transcription reagent instructions (Vazyme).

[0069] 1)Thaw the template RNA on ice; thaw the reagent kit components at room temperature immediately on ice. Before use, vortex each solution to mix well, and use after a short centrifugation.

[0070] 2)According to the following table, prepare the reaction system on ice, with a total volume of 20 μl (see Table 1). In order to ensure the accuracy of the reaction liquid preparation, first prepare the premixed reaction system according to the reaction number + 2, then dispense into each reaction tube, and finally add the RNA sample.

[0071] 3)Vortex to mix well, and centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.

[0072] 4)Incubate at 50℃ for 5 min, and at 85℃ for 5 sec.

[0073] 5)After the reaction is completed, centrifuge briefly and cool on ice.

[0074] Table 1 Reverse transcription reaction system

[0075]

[0076] Three-step qPCR, according to the qPCR reagent instructions (Vazyme):

[0077] 1)Thaw the template DNA on ice, dilute 4 times with water before use; dry powder of upstream and downstream primers at 4℃, centrifuge at 12000 rpm for 1 min, then add water according to the primer tube label, and calculate 1:1 premixed upstream and downstream primers according to the use volume (see Table 2). Set the internal reference gene (GAPDH) and blank control, and vortex each solution to mix well before use, and use after a short centrifugation.

[0078] Table 2 Primer sequence

[0079]

[0080] 2)According to the following table, prepare the reaction system on ice, with a total volume of 20 μl (see Table 3), and at least 3 replicate wells are recommended.

[0081] 3) Mix well and centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.

[0082] 4) qPCR reaction conditions: Instrument model: ABI7500 Fast.

[0083] 5) The reaction conditions are 95℃ for 30s; 95℃ for 10s and 40 cycles; and 60℃ for 30s.

[0084] Table 3 qPCR reaction system

[0085]

[0086] Data processing and experimental results

[0087] First, use statistical analysis software (Excel, Graphpad, etc.) to calculate the mean Ct of each test sample; then calculate the ∆Ct of the test sample, which is the Ct of a certain test factor in a certain test sample minus the Ct of the internal reference gene in a certain test sample; then calculate the ∆∆Ct of the test sample, which is the ∆Ct of the experimental group of a certain test factor minus the ∆Ct of the blank group of a certain test factor; finally, calculate the 2^(-∆∆Ct) value of the test sample, which is the relative expression level, and the standard error (SD) of each test sample well to obtain the data table.

[0088] The results are as follows Figure 7 As shown, humanized collagen type XVII peptide A has the effect of promoting the upregulation of β-Catenin signaling pathway protein expression, and is superior to competitor product 1 (Recol 17). ® (Purchased from Jiangsu Chuangjian Medical Technology Co., Ltd.) The results show that humanized collagen type XVII polypeptide A has good anti-hair loss and hair growth effects.

[0089] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A humanized collagen type XVII polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A polynucleotide encoding the polypeptide of claim 1.

3. The polynucleotide according to claim 2, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.

2.

4. An expression carrier, characterized in that, The expression vector comprises the polynucleotide as described in claim 2 or 3.

5. A host cell, characterized in that, The host cell comprises the expression vector of claim 4.

6. The host cell according to claim 5, characterized in that, The host cell is an Escherichia coli cell.

7. A method for preparing the polypeptide according to claim 1, characterized in that, The preparation method includes: 1) culturing the host cells of claim 5 or 6 in a production culture medium; and 2) isolating the polypeptide from the host cells.

8. A composition having anti-hair loss and hair growth functions and / or skin repair, anti-wrinkle and firming functions, characterized in that, The composition comprises the polypeptide of claim 1.

9. A protein product with anti-hair loss and hair growth functions and / or skin repair, anti-wrinkle and firming functions, characterized in that, The protein product includes the polypeptide of claim 1 or the composition of claim 8, and the protein product is a tissue-engineered product or a cosmetic.

10. The use of the polypeptide of claim 1, or the polynucleotide of claim 2 or 3, or the expression vector of claim 4, or the host cell of claim 5 or 6, or the composition of claim 8 in the preparation of protein products having anti-hair loss and hair growth functions and / or skin repair, anti-wrinkle and firming functions, characterized in that, The protein products mentioned are tissue-engineered products or cosmetics.

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