Recombinant humanized 17-type collagen mussel mucin polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a recombinant humanized 17-type collagen mussel mucin polypeptide as well as a preparation method and application thereof, and belongs to the technical field of genetic engineering. According to the invention, fragments of human-derived 17-type collagen and histidine and 3, 4-dihydroxyphenylalanine rich regions of mussel mucin are recombined, and the obtained recombinant protein has higher cell adhesion activity. The amino acid sequence of the polypeptide is as shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4. The recombinant humanized 17-type collagen mussel mucin polypeptide can be expressed at a relatively high level by taking escherichia coli as a host, is simple in preparation method and easy for expanded production, and can be widely applied to industries such as medicines and cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a recombinant human type 17 collagen mussel mucin polypeptide and a preparation method and application thereof. Background Art

[0002] Hair loss is one of the skin diseases that plague the health of modern people and can seriously affect the mental health of patients. As people's pace of life continues to accelerate and the pressure of life and work increases, hair loss has become a common problem in society and is showing a trend of younger age. Human hair growth goes through three stages: growth, decline and dormancy, which are mainly affected by the state of hair follicle stem cells. Hair follicle stem cells are located in the bulge / subbulge area of ​​the hair follicle and maintain the cyclic regeneration of hair in the repetitive hair cycle. With age, hair follicle stem cells will enter a decline period, and their aging will lead to the gradual miniaturization of hair follicles in wild-type mice and humans, and eventually cause hair loss. The mechanism of hair loss is relatively complex. Early studies have found that the hydrolysis of type XVII collagen (COL17A1) in hair follicle stem cells is an important factor in triggering the aging of hair follicle stem cells and the miniaturization of hair follicles (Matsumura, H., et.al, 2016. Hair follicle aging is driven by transepidermalelimination of stem cells via COL17A1 proteolysis. Science 351, aad4395). COL17A1 is usually distributed in large quantities throughout the cell surface and cytoplasm of young keratinocytes. Researchers have found that the content of COL17A1 is significantly reduced in some miniaturized hair follicles. Matsumura et al. analyzed the miniaturization of hair follicles and the expression of COL17A1 in women aged 22 to 70 years. The scalp of human women aged 55-70 years contained significantly more small hair follicles, and the expression of COL17A1 was significantly reduced only in small hair follicles, but not in non-small hair follicles, even in the skin of the elderly scalp. Further studies found that forcibly maintaining COL17A1 in hair follicle stem cells can prevent their aging, indicating that COL17A1 plays an important role in hair follicle stem cell differentiation and hair follicle miniaturization. Recent studies have shown that type 17 collagen not only plays an important role in maintaining skin elasticity, protecting joints and connective tissue health, but also has the potential to promote skin repair and hair regeneration. Type 17 collagen can increase the proliferation and differentiation of hair follicle matrix cells, improve the biological activity of hair follicles, promote hair growth, and treat hair loss (Zheng Yongyi, Liu Qin, Yang Chaoru, et al. Clinical efficacy of type 17 collagen in the treatment of androgenic alopecia [J]. Journal of Wuhan University (Medical Edition), 1-5). The content of type 17 collagen in animals is extremely low. The collagen extracted from animals has poor water solubility and poor processability, which directly limits the development of many potential uses. At present, the preparation of type 17 collagen through biotechnology such as genetic engineering can effectively overcome the above problems.

[0003] Recombinant mussel mucin is an artificial protein obtained by genetic recombination of marine mussels. It has broad application prospects due to its good adhesion, degradability, flexibility and water resistance. The three main functions of recombinant mussel mucin are biological adhesion, formation of protective film and promotion of cell adhesion and crawling. It has the effect of promoting wound healing in clinical practice. Recombinant mussel mucin is a protein with a high content of dopa groups. The dopa group has obvious anti-inflammatory and antioxidant activities, which can promote the formation of a protective layer on the skin surface and improve sensitive skin problems. In addition, mussel mucin is mild in nature, non-irritating, and has good safety and tolerance. At present, mussel mucin has been widely used in medical beauty fields such as ophthalmic surgery, skin tissue adhesion and bone adhesion as a medical adhesive. In recent years, researchers have used genetic engineering technology to recombinantly express mussel mucin, but the adhesion performance of the recombinant protein is far inferior to that of natural mussels, which limits its in-depth research and practical application. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a recombinant human type 17 collagen mussel mucin polypeptide. The recombinant human type 17 collagen mussel mucin polypeptide provided by the present invention has higher cell adhesion activity.

[0005] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0006] A recombinant human type 17 collagen mussel mucin polypeptide having an amino acid sequence selected from the following A) or B):

[0007] A) the amino acid sequence shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4;

[0008] B) A derivative sequence based on the amino acid sequence shown in A) by substitution, deletion, replacement, insertion and / or addition of one or more amino acid residues.

[0009] As a preferred embodiment of the present invention, the polypeptide is a polypeptide composed of a derivative sequence of the amino acid sequence shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4, which is substituted, deleted, replaced, inserted and / or added with one or more amino acid residues and has protein activity.

[0010] As a preferred embodiment of the present invention, the polypeptide includes a type 17 collagen fragment and a mussel mucin fragment, and the type 17 collagen fragment sequence is connected to the mussel mucin fragment sequence through a peptide bond. Wherein, the type 17 collagen fragment sequence is derived from human type 17 collagen; the mussel mucin fragment sequence is derived from mussel mucin sequences such as Mytiluscalifornianus, Mytiluscalifornianus, Mytilusedulis, Mtiluscoruscus, and Mytilusgalloprovincialis.

[0011] A second object of the present invention is to provide an isolated polynucleotide encoding the polypeptide as described above.

[0012] As a preferred embodiment of the present invention, the nucleotide sequence of the polynucleotide is selected from the nucleotide sequence shown in any one of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8.

[0013] A third object of the present invention is to provide a recombinant expression vector, which comprises the polynucleotide as described above.

[0014] A fourth object of the present invention is to provide a host cell, wherein the host cell has the function of expressing the polypeptide as described above; or the host cell contains the polynucleotide or recombinant expression vector as described above.

[0015] In some embodiments of the invention, the host cell comprises a bacterial or fungal cell.

[0016] As a preferred embodiment of the present invention, the host cell is an Escherichia coli cell.

[0017] A fifth object of the present invention is to provide a method for preparing the polypeptide as described above, which comprises the following steps:

[0018] (1) culturing the host cell as described above in a production medium;

[0019] (2) Isolating the polypeptide as described above from the host cell.

[0020] More specifically, the preparation method comprises the following steps:

[0021] (1) culturing the aforementioned host cells under appropriate culture conditions;

[0022] (2) inducing the expression of the target gene in the host cell in step (1) to obtain a recombinant human type 17 collagen mussel mucin polypeptide;

[0023] (3) Harvesting the host cells and / or culture medium containing the recombinant human type 17 collagen mussel mucin polypeptide, and isolating and purifying the recombinant collagen mussel protein polypeptide in step 2).

[0024] The sixth object of the present invention is to provide a composition comprising the recombinant human type 17 collagen mussel mucin polypeptide as described above.

[0025] The seventh object of the present invention is to provide a product, which comprises the recombinant human type 17 collagen mussel mucin polypeptide or composition as described above; wherein the product is a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic or a health product.

[0026] The seventh object of the present invention is to provide the use of the recombinant human type 17 collagen mussel mucin polypeptide, polynucleotide, recombinant expression vector, host cell or composition as described above in the preparation of a finished product, wherein the finished product is a medical device, a tissue engineering product, a cosmetic or a health product.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention combines the histidine-rich region and 3,4-dihydroxyphenylalanine (DOPA)-rich region in the human type 17 collagen sequence and the mussel mucin sequence for the first time, and recombinantly synthesizes a new protein material to obtain a recombinant human type 17 collagen mussel mucin polypeptide. Compared with single collagen and mussel mucin, the recombinant protein has higher cell adhesion. The recombinant type 17 collagen mussel protein gene contains the characteristics of both human collagen and mussel mucin genes, and the protein expressed by it will have the excellent characteristics of natural human collagen and natural mussel mucin. The recombinant human type 17 collagen mussel mucin polypeptide of the present invention can be produced in Escherichia coli, and high-density fermentation can achieve a higher level of expression of recombinant type 17 collagen mussel mucin. The preparation method is simple and easy to scale up. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The plasmid map of the vector pET32a-CO17AMFBERI1 of the present invention;

[0030] Figure 2 The plasmid map of the vector pET28a-CO17AMFBERI2 of the present invention;

[0031] Figure 3 The plasmid map of the vector pET32a-CO17AMFBERI3 of the present invention;

[0032] Figure 4 The plasmid map of the vector pET24a-CO17AMFBERI4 of the present invention;

[0033] Figure 5 This is the electrophoresis diagram of the target proteins CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3 and CO17AMFBERI4 expressed in Escherichia coli of the present invention; the electrophoresis detected molecular weights of CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3 and CO17AMFBERI4 proteins are approximately 37KDa, 28KDa, 35KDa and 57kDa, respectively.

[0034] Figure 6 This is the protein electrophoresis diagram obtained by expressing and purifying the CO17AMFBERI1, CO17AMFBERI2 and CO17AMFBERI3 proteins of the present invention; the protein electrophoresis detected molecular weights were approximately 37KDa, 28KDa and 35KDa.

[0035] Figure 7 The results of biological activity test of CO17AMFBERI1, CO17AMFBERI2 and CO17AMFBERI3 proteins of the present invention compared with type 17 human collagen and mussel mucin. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the following embodiments do not limit the protection scope of the present invention.

[0037] Unless otherwise specified, the biological materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or by existing known methods. The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Laboratory Manual (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0038] The test materials and reagents used in the following examples are as follows:

[0039] 1. Strains, cells and vectors

[0040] Strains containing CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3, CO17AMFBERI4 genes and expression plasmids, Escherichia coli DH5a, TOP10, BL21DE3, Rosetta (DE3), BL21 (DE3) PLysE, BL21 (DE3) PLysS and other strains were purchased from Biyuntian and Pichia pastoris X33 (Miaoling Biotechnology), and vectors pet32a, pet28a, and pet24 were purchased from Invitrogen.

[0041] 2. Kits and enzymes

[0042] LDH detection kit (Roche 04744926001), modified Bradford protein content assay kit (Sangon Biotechnology), seamless cloning kit In-Fusion Snap Assembly Master Mix (TaKaRa), plasmid extraction kit (Universal Biotechnology), gel recovery kit (Universal Biotechnology), restriction endonuclease, Q5 ultra-fidelity 2x Master Mix PCR polymerase, etc. were purchased from NEB.

[0043] 3. Culture medium

[0044] The culture medium for E. coli was LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0), LB+AMP medium was LB medium with ampicillin added to a final concentration of 100 μg / ml, LB+Kana medium was LB medium with kanamycin added to a final concentration of 25 μg / ml, and LB+Zeo was LB medium with Zeocin added to 100 μg / ml;

[0045] Escherichia coli induction medium and high-density fermentation medium, the large intestine induction medium is TB medium (2% yeast powder, 1% peptone, 1.5% potassium dihydrogen phosphate, 2% potassium dihydrogen phosphate, adjusted to pH 7.0), the inducer is 0.2-0.4mM IPTG; the large intestine high-density fermentation medium is TB based with 0.1%-0.2% defoamer added, the pH is 7.0, and the feed glycerol is 400g / L;

[0046] 4. Electrophoresis reagents

[0047] SDS-PAGE loading buffer (BioSharp), PageRuler TM Plus pre-stained protein 10-250kDa (Thermo Fisher) Coomassie Brilliant Blue staining solution (0.25% Coomassie Brilliant Blue R-250, 45% methanol (v / v), 10% glacial acetic acid (v / v)), decolorization solution (10% methanol (v / v), 10% glacial acetic acid (v / v)).

[0048] 5. Protein purification materials

[0049] Ni fillers and nickel columns were purchased from Sanji Biotechnology.

[0050] In the present invention, the recombinant protein can be produced by conventional methods in the art. For example, it can be produced by the following steps: (1) construction of genetically engineered Escherichia coli; (2) fermentation culture of genetically engineered Escherichia coli; (3) induction and expression of recombinant type 17 collagen mussel protein; and (4) purification of recombinant type 17 collagen mussel protein.

[0051] Example 1: Gene synthesis of recombinant type 17 collagen mussel mucin polypeptide

[0052] Gene analysis: The amino acid sequence of natural human type 17 collagen and the fp6, fp5, fp4, fp3, and fp1 protein sequences from Mytilus californianus, Mytilus californianus, Mytilus edulis, Mtilus coruscus, and Mytilusgalloprovincialis were selected for analysis of physical and chemical properties such as hydrophilicity and isoelectric point, and the amino acid sequences with strong water solubility, histidine-rich regions, and DOPA-rich regions were selected for recombination. The recombinant sequences were codon-optimized for Escherichia coli codons, and the new genes obtained by recombination were the genes CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3, and CO17AMFBERI4 of the present invention, and the amino acid sequences were as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

[0053] Gene synthesis: The full lengths of CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3, and CO17AMFBERI4 genes are 594 bp, 768 bp, 600 bp, and 1548 bp, respectively, and the sequences after codon optimization are SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively. The optimized sequences were subjected to gene synthesis, and the gene fragments were provided by General Gene Biotechnology Co., Ltd., and the synthesized CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3, and CO17AMFBERI4 gene fragments were inserted into the expression vectors of PET32a, PET28a, or PET24a through the restriction sites of Kpn I and Xho I or BamHI and HindIII, respectively, and then the vectors were transformed into Escherichia coli TOP10 or DH5a for preservation.

[0054] The recombinant expression vector maps of the four genes CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3 and CO17AMFBERI4 are shown in Figures 1 to 4 .

[0055] Example 2: Host bacteria construction and induced expression of recombinant human type 17 collagen mussel mucin polypeptide

[0056] Plasmid extraction: Take the synthesized CO17AMFBERI1 / TOP10, CO17AMFBERI2 / TOP10, CO17AMFBERI3 / TOP10, CO17AMFBERI4 / TOP10 and streak the corresponding resistance on LB+A or LB+K plates, and culture them at 37℃ overnight. Pick the single clones cultured overnight and inoculate them into 10ml LB+A or LB+K liquid medium, and culture them at 37℃ overnight. Take the bacterial solution cultured overnight, centrifuge at 5000rpm for 5min, discard the supernatant and take the bacteria. Use Tiangen Plasmid Extraction Kit to extract the plasmid. After the extracted plasmid is measured by NanoDrop, it can be directly transformed into the expression host or frozen at -20℃.

[0057] Strain construction: Take 2ul of CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3, CO17AMFBERI4 plasmids and put them in 100μl of E. coli competent cells, and let them stand on ice for 15-30min. Heat shock the mixture in a 42℃ water bath for 45s, let it stand on ice for 2-3min, add 500μl of LB liquid medium without resistance, and resuscitate and culture at 37℃ and 190rpm for 20-45min. Take 100μl of the bacterial solution and evenly spread it on LB+K or LB+Amp plates. Culture it at 37℃ overnight until clearly visible colonies grow.

[0058] Induced expression of target protein: From the overnight culture plate, pick a single clone and culture it in 10ml LB+A or LB+K liquid medium overnight. Then transfer it to TB medium at a ratio of 1% for expansion culture. When the bacterial solution OD600 is 0.5-0.8 at 37℃ and 190rpm, add IPTG with a final concentration of 0.1-0.5mM to induce expression. The induction conditions are 16-26℃ and 190rpm for 16-20h. Collect the bacteria by centrifugation and wash them repeatedly with phosphate buffer for 3 times. The washed bacteria can be frozen at minus 20℃ or directly broken by high pressure sterilization instrument. The broken mixture is centrifuged at 10000rpm for 30min, and the supernatant after centrifugation is taken for storage.

[0059] SDS-PAGE detection of target protein: Take 20μl of the supernatant after centrifugation, add 10μl of 3× protein loading buffer, place in 100℃ boiling water for 10min, then add 20μl to each well into the SDS-PAGE protein gel, first run at 80V voltage for 1h, then turn to 120V and run until the bands are completely separated. Use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R 250, 25% isopropanol, 10% glacial acetic acid) to stain the protein for 30min, and then use protein decolorizing solution (10% acetic acid, 5% ethanol) to decolorize overnight. The electrophoresis results are shown in Figure 5 .

[0060] like Figure 5 As shown, the electrophoresis detected molecular weights of the proteins of the four genes CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3, and CO17AMFBERI4 were approximately 37KDa, 28KDa, 37KDa, and 62kDa, respectively.

[0061] Example 3: Purification of recombinant human type 17 collagen mussel mucin polypeptide

[0062] Crude protein treatment: Take the frozen cells washed with phosphate buffer, weigh them, resuspend them with 8 times the volume of lysis solution (50mM PB, 10mM imidazole, 0.5M NaCl, pH7.0), and use a high-pressure homogenizer to break them under low temperature conditions. The mixed solution after breaking is centrifuged at 4°C, 10000rpm, 25min, discard the precipitate, harvest the supernatant, and calculate the volume.

[0063] Ni-NTA nickel column protein purification: Use (50mM PB, 0.5M NaCl, pH7.0) to rinse 5 column volumes to balance the nickel column: Take the pretreated protein for loading. Rinse 3-5 column volumes with low concentration imidazole (10-50mM) buffer to remove impurities, and then gradient elute with 50-500mM imidazole. Collect protein samples eluted with different concentrations of imidazole, and detect purity by SDS-PAGE. After collection, rinse the nickel column with 20% ethanol and store it after filling with ethanol. Take the protein purification solution received at different times and perform SDS-PAGE detection to determine the purity and size of the target protein. The nickel column that has been used repeatedly needs to be regenerated. The regeneration steps are as follows: rinse 5 column volumes with ddH2O, rinse 5 column volumes with EDTA solution, rinse 5 column volumes with NaOH solution, fill the affinity column with NiSO4 solution, and rinse 5 column volumes.

[0064] like Figure 6As shown, the electrophoresis detected molecular weights of the proteins of the three genes CO17AMFBERI1, CO17AMFBERI2, and CO17AMFBERI3 were approximately 37KDa, 28KDa, and 35KDa, respectively.

[0065] Example 4: Detection of cell adhesion activity of recombinant human type 17 collagen mussel mucin polypeptide

[0066] The activity of the recombinant protein is detected by detecting the effect of the protein on the cell adhesion activity. The specific detection method can be slightly modified by referring to patent CN 110845603 A and the literature Yao et al. 2004. Design, Expression and Characterization of Collagen Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649). The specific process is as follows:

[0067] Protein content determination: Use the modified Bradford protein concentration determination kit to detect protein content. Take the sample dilution, add 1mL Bradford working solution to each tube, mix quickly, react at room temperature 25-30℃ for 10min, use tube 0 as blank control, and measure the A595 value of each tube on the spectrophotometer. After the protein concentration is detected, adjust the concentration of all proteins to be tested to 0.5mg / ml with PBS, filter and sterilize with a 0.22μm microporous filter membrane, and set aside.

[0068] 3T3 cell culture: 3T3 mouse embryonic fibroblasts were cultured in DMEM containing 1% G / A and 10% fetal bovine serum. The specific process is as follows: 3T3 cells were quickly taken out of liquid nitrogen and placed in a 37°C water bath for thawing, followed by adding 2 mL of culture medium, centrifuging at 800 rpm for 3 min, discarding the supernatant, adding 1 mL of culture medium to resuspend the cells and transferring them to a T25 culture flask, adding 4 mL of culture medium, and culturing in a 37°C, 5% CO2 incubator for 1 to 2 days. Subculture was performed when the cell fusion degree was about 85%. The cells were washed with PBS, and then 1 mL of 0.25% trypsin was added to digest the attached cells, and then two times the volume of complete culture medium was added to terminate the digestion, centrifuged at 800 rpm / min for 5 min, discarding the supernatant, and adding culture medium to resuspend the cells. The cell suspension was transferred to two T25 culture flasks, and 4 mL of culture medium was added to continue culturing the cells. 5000 3T3 cells in good growth state were seeded into a 96-well plate at 100 μL per well, and 100 μL PBS buffer was added to the edge wells, and incubated in an incubator for 24 hours before use.

[0069] Cell adhesion activity detection: PBS was used as negative control, bovine serum albumin, human collagen and mussel mucin were used as positive controls, different recombinant proteins were used as test samples, and the culture medium without cells was used as zero well. 100 μL of the corresponding group solution was added to the 96-well ELISA plate, with 3 replicates per group, and incubated overnight in a 4°C refrigerator. At the end of incubation, the excess coating solution was aspirated from the wells, washed 3 times with PBS, and the washing solution was discarded. The pre-cultured 3T3 cells were inoculated in a 96-well plate at 150 μL per well, and 100,000 were inoculated in a 37°C cell culture incubator for 2 hours. At the end of incubation, the supernatant was discarded and washed 3 times with PBS. The cell adhesion was detected using the LDH detection kit and detected at a wavelength of 492 nm on an ELISA reader.

[0070] Results Figure 7 , Figure 7 The results showed that four recombinant human type 17 collagen mussel mucins (CO17AMFBERI1, CO17AMFBERI2, CO17AMFBERI3) all had better cell adhesion activity compared with commercial human collagen and mussel mucins.

[0071] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, any insubstantial changes and replacements made on the basis of the present invention for those skilled in the art as described above should be included within the scope of the present invention.

Claims

1. Recombinant human type 17 collagen mussel mucin polypeptide, characterized in that: The polypeptide has an amino acid sequence selected from the following A) or B): A) the amino acid sequence shown in any one of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4; B) A derivative sequence based on the amino acid sequence shown in A) by substitution, deletion, replacement, insertion and / or addition of one or more amino acid residues.

2. A polynucleotide encoding the polypeptide according to claim 1.

3. The polynucleotide according to claim 2, characterized in that: The nucleotide sequence of the polynucleotide is selected from the nucleotide sequence shown in any one of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 and SEQ ID No.

8.

4. A recombinant expression vector, characterized in that: Comprising the polynucleotide according to claim 2 or 3.

5. A host cell, characterized in that: It comprises the polypeptide according to claim 1, the polynucleotide according to claim 2 or 3, or the recombinant expression vector according to claim 4.

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

7. The method for preparing a polypeptide according to claim 1, characterized in that: The following steps are involved: (1) culturing the host cell according to claim 5 in a production medium; (2) Isolating the polypeptide according to claim 1 from the host cell.

8. A composition, characterized in that: Comprising the polypeptide according to claim 1.

9. A product, characterized in that: Comprising the polypeptide according to claim 1 or the composition according to claim 8; wherein the product is a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic or a health product.

10. Use of the polypeptide according to claim 1, the polynucleotide according to claim 3, the expression vector according to claim 4, the host cell according to claim 5 or 6, or the composition according to claim 8 in preparing a finished product, characterized in that: The finished product is a medical device, a tissue engineering product, a cosmetic or a health product.

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