Self-assembled super-elastic protein, hydrogel and application thereof

Abstract

The application discloses a kind of self-assembly super elastic protein, hydrogel and its application, belong to the field of biotechnology.The self-assembly super elastic protein of the application includes: self-assembly peptide SUP35, human-derived elastin and human-derived adhesive peptide;The amino acid sequence of the self-assembly peptide SUP35 is as shown in SEQ ID NO.3;The amino acid sequence of the human-derived elastin is as shown in SEQ ID NO.4;The sequence of the human-derived adhesive peptide is VAPG;The number of the human-derived elastin is 1 or more.The self-assembly super elastic protein of the application is obtained by fermentation, purification using microorganism as the bottom disc, reduces a series of problems caused by source problem.Simultaneously super elastic protein can be self-assembled to form multistage structure, has the function of significant high-temperature resistance, cell proliferation, cell migration, cell repair, and has significant antioxidant capacity and excellent in-vitro adhesion.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a self-assembled superelastic protein, hydrogel, and their applications. Background Technology

[0002] Elastin is a naturally occurring extracellular matrix protein, renowned for its excellent elasticity, mechanical stability, and biocompatibility. Natural elastin is primarily derived from animal tissues (such as bovine cervical ligaments and porcine aorta), but this process is limited by factors such as restricted sources, complex extraction procedures, and poor batch-to-batch stability. Furthermore, animal-derived components are prone to immune rejection and pathogen contamination, making direct application to human tissue repair difficult. While recombinant elastin or its fragments prepared through genetic engineering have solved the biocompatibility issue, they lack the complex multi-level structure of natural elastin, resulting in insufficient mechanical properties and a tendency for structural collapse or functional failure in dynamic mechanical environments. To improve the stability or mechanical properties of elastin, current technologies often employ chemical cross-linking (such as glutaraldehyde and genipin). However, residual cross-linking agents can reduce biocompatibility, and excessive cross-linking can lead to increased material brittleness and uncontrolled degradation rates.

[0003] Peptide molecules self-assemble into ordered supramolecular structures through non-covalent interactions such as hydrogen bonds, hydrophobic interactions, and van der Waals forces. The synergistic accumulation of these weak interactions maintains the dynamic stability of the structure and fosters complex functional properties. However, the assembly behavior of most existing self-assembly materials is highly dependent on environmental factors (temperature, pH, ionic strength). Even small fluctuations can lead to non-uniformity in the assembled structure (such as fiber diameter and pore size distribution), resulting in poor material repeatability and making standardized production difficult. While natural materials (such as collagen and hyaluronic acid) have good biocompatibility, they have low mechanical strength, are not resistant to high temperatures, and degrade too quickly, failing to meet the repair needs of weight-bearing tissues. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide a self-assembled superelastic protein, hydrogel, and its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a self-assembled superelastic protein, comprising: a self-assembled peptide SUP35, human elastin, and a human adhesion peptide;

[0007] The amino acid sequence of the self-assembled peptide SUP35 is shown in SEQ ID NO.3; the amino acid sequence of the human elastin is shown in SEQ ID NO.4; and the sequence of the human adhesion peptide is VAPG.

[0008] The human elastin is composed of a hydrophilic sequence (AAAAAKAAAKAAK), a hydrophobic sequence (VGVPGAGVPGVGVPGAG), and a hydrophilic sequence (AAAAAKAAAKAAK), forming a 3-block copolymer; the number of human elastin is one or more repeats, preferably one.

[0009] In some preferred embodiments of the present invention, the amino acid sequence of the self-assembled hyperelastic protein is shown in SEQ ID NO. 6.

[0010] In some preferred embodiments of the present invention, the self-assembled hyperelastic protein is prepared by the following method:

[0011] (1) Construct a recombinant plasmid containing the gene encoding self-assembled superelastic protein, and transfer the recombinant plasmid into competent Escherichia coli cells to obtain recombinant bacteria;

[0012] (2) Inoculate the recombinant bacteria into LB liquid medium and culture until OD. 600nm When the concentration is 0.3-0.6, IPTG is added for induction culture; the induced culture solution is centrifuged, the bacterial cells are collected, sonicated and disrupted, centrifuged again, the supernatant is collected and purified to obtain self-assembled superelastic protein.

[0013] Preferably, in step (1), the nucleotide sequence of the self-assembled hyperelastic protein encoding gene is shown in SEQ ID NO.5.

[0014] Preferably, in step (2), the final concentration of IPTG added is 0.5mM, and the induction culture conditions are: 37℃, 180rpm constant temperature shaker culture for 4h.

[0015] In a second aspect, the present invention provides a hydrogel obtained by the self-assembly of the above-described self-assembled superelastic protein in a solvent;

[0016] The solvent is pure water, PBS buffer, TB buffer, cell culture medium, or bacterial culture medium.

[0017] Preferably, the concentration of the self-assembled hyperelastic protein in the solvent is not less than 0.7 mg / mL.

[0018] A third aspect of the present invention provides the application of the above-described self-assembled superelastic protein or hydrogel in the preparation of adhesive materials.

[0019] The self-assembled superelastic protein or hydrogel of the present invention has excellent in vitro adhesion properties and can be used to prepare adhesive materials.

[0020] In a fourth aspect, the present invention provides the use of the above-described self-assembled superelastic protein or hydrogel in the preparation of a culture medium that promotes cell proliferation, cell migration and / or cell damage repair.

[0021] A fifth aspect of the present invention provides the use of the above-described self-assembled superelastic protein or hydrogel in the preparation of a free radical scavenger, wherein the free radical is DPPH, ·OH or ABTS.

[0022] The beneficial effects of this invention are:

[0023] (1) The self-assembled superelastic protein of the present invention is composed of self-assembling peptide SUP35, human elastin and human adhesion peptide, and has excellent gelling properties with a gelling concentration as low as 0.7 mg / mL; it can gel after a short period of standing treatment under conditions higher than the gelling concentration. More importantly, the self-assembled superelastic protein of the present invention also has significant high temperature resistance, can withstand high temperatures of 100°C, and can still self-assemble into a gel structure after high temperature treatment.

[0024] (2) The self-assembled superelastic protein of the present invention is obtained by fermentation and purification using microorganisms as a chassis, which reduces a series of problems caused by source issues. At the same time, the prepared self-assembled superelastic protein also has the functions of promoting cell proliferation, cell migration, and cell repair, as well as antioxidant capacity and in vitro adhesion, and has a wide range of application value. Attached Figure Description

[0025] Figure 1 FuzDrop analysis results of Sup35 prion protein in Saccharomyces cerevisiae.

[0026] Figure 2 Results of IUPred2 analysis of Saccharomyces cerevisiae Sup35 prion protein.

[0027] Figure 3 PLAAC platform analysis results of Saccharomyces cerevisiae Sup35 prion protein.

[0028] Figure 4 : Schematic diagram of the structure of the recombinant plasmid constructed in Example 3.

[0029] Figure 5 Example 3 shows the SDS-PAGE results of the self-assembled superelastic protein before and after purification. In the figure, A is the SDS-PAGE image before purification, M: Marker, 1: SUPELN fermentation broth; 2: SUPELN ultrasonically disrupted supernatant; 3: SUPELN ultrasonically disrupted precipitate. B is the SDS-PAGE image after purification, M: Marker, 1-4 are all purified and desalted proteins.

[0030] Figure 6 Low-voltage transmission electron microscopy images of the self-assembled superelastic protein prepared in Example 3 at different magnifications; in the figures, the scale bar of A is 500 nm; the scale bar of B is 200 nm.

[0031] Figure 7 The results of the high-temperature resistance test of the self-assembled superelastic protein of the present invention.

[0032] Figure 8 Images taken at 0 and 24 hours after cell scratch treatment; In the figures, A represents treatment with 0.25 mg / mL self-assembled hyperelastin; B represents treatment with 0.25 mg / mL commercially available collagen; C represents treatment with 0.125 mg / mL self-assembled hyperelastin; D represents treatment with 0.125 mg / mL commercially available collagen; E represents treatment with 0.0625 mg / mL self-assembled hyperelastin; F represents treatment with 0.0625 mg / mL commercially available collagen; G represents treatment with 0.03125 mg / mL self-assembled hyperelastin; H represents treatment with 0.03125 mg / mL commercially available collagen.

[0033] Figure 9 Results of cell scratch migration assay.

[0034] Figure 10 The results of the self-assembled superelastic protein of the present invention on the adhesion of BALB / c 3T3 cells.

[0035] Figure 11 Experimental results on injury prevention.

[0036] Figure 12 : Experimental results of intervention for injury.

[0037] Figure 13 Results of ·OH scavenging capacity assay of self-assembled superelastic protein.

[0038] Figure 14 Results of ABTS scavenging ability assay of self-assembled hyperelastin; In the figure, A represents the ABTS free radical scavenging results at self-assembled hyperelastin concentrations of 10, 5, 3, 1, and 0.5 mg / mL; B represents the ABTS free radical scavenging results at self-assembled hyperelastin concentrations of 0.5, 0.3, 0.1, 0.05, 0.01, and 0.005 mg / mL; C represents the ABTS free radical scavenging results at self-assembled hyperelastin concentrations of 0.1, 0.05, 0.025, 0.01, 0.005, and 0.0025 mg / mL.

[0039] Figure 15 Results of the weight adhesion test. Detailed Implementation

[0040] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0041] As mentioned earlier, most existing self-assembling elastins are highly dependent on environmental factors (temperature, pH, ionic strength). Even small fluctuations can lead to uneven assembly structures (such as fiber diameter and pore size distribution), poor material repeatability, and difficulty in achieving standardized production.

[0042] Therefore, this invention first uses the Sup35 prion protein of Saccharomyces cerevisiae as the research object, and selects the N-terminal 1-240AA region of the Sup35 prion protein of Saccharomyces cerevisiae as the candidate region for self-assembled peptides by performing FuzDrop analysis, IUPred2 analysis and PLAAC analysis.

[0043] Then, three peptide segments of different lengths—1-61AA (SEQ ID NO.2), 1-200AA (SEQ ID NO.3), and 1-240AA (SEQ ID NO.1)—were selected from the N-terminal 1-240AA region of the Saccharomyces cerevisiae Sup35 prion protein to examine their gel-forming properties. The results showed that the 1-200AA (SEQ ID NO.3) peptide was suitable for prokaryotic expression and had the best gel-forming properties. Therefore, 1-200AA (SEQ ID NO.3) was selected as the self-assembling peptide SUP35.

[0044] The self-assembled peptide SUP35 was then fused with different elastin fragments. It was found that the fusion effect of the self-assembled peptide SUP35 with the human elastin shown in SEQ ID NO.4 was the best. In order to balance the adhesion properties of the self-assembled elastin, a human adhesion peptide (VAPG) was added to the end, and finally a new self-assembled superelastic protein SUPELN was prepared, the amino acid sequence of which is shown in SEQ ID NO.6.

[0045] The self-assembled superelastic protein of the present invention has the following many excellent properties:

[0046] (1) The gel concentration is low, the gelation time is short, and the gelation conditions are simple. It can be gelled by standing.

[0047] (2) It has high temperature resistance. The self-assembled superelastic protein can still be stably gelled after high temperature treatment.

[0048] (3) It has strong self-assembly ability, excellent superelasticity, and dense and stable network. The self-assembly peptide SUP35, elastin and human adhesion peptide can synergistically improve the performance of the self-assembled superelastic protein.

[0049] (4) It has the functions of promoting cell proliferation, cell migration, and cell repair, as well as antioxidant capacity.

[0050] (5) It has excellent in vitro adhesion ability.

[0051] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0052] The test materials used in the embodiments and comparative examples of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions were performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0053] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0054] LB solid medium: Add 15g of agar powder to each liter of LB liquid medium.

[0055] Commercially available recombinant type III humanized collagen, sourced from Jinbo Biotechnology, was used. The BCA protein concentration assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. BALB / c 3T3 cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd. DMEM high-glucose medium was purchased from Gibco (catalog number: C11995500BT). The CCK-8 assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0056] The pH of the PBS used was 7.2-7.4.

[0057] Example 1: Biological prediction analysis of the self-assembled peptide fragments of Sup35 prion protein in Saccharomyces cerevisiae

[0058] 1. FuzDrop analysis:

[0059] The full-length sequence of the Sup35 prion protein (NP_010457.3) from *Saccharomyces cerevisiae* was predicted using the FuzDrop platform, and the results are as follows: Figure 1 As shown, the results indicate that pLLPS = 0.6313 (medium to high), suggesting that it readily undergoes LLPS (liquid-liquid phase separation) even without a partner. The driving force mainly comes from the N / M disordered regions (1-101, 108-241), which also contain aggregation hotspots (62-74, 84-101, 108-142), making the droplets prone to gelation / fibrillation. The C-terminus has weak overall droplet-forming ability, but a few small peaks can participate under specific conditions. Multiple context-dependent segments (such as 62-74, 84-104, 116-142, 318-333, 546-568, 571-594, 638-650) indicate that its phase behavior is highly sensitive to salt, pH, crowding, and modification. This suggests that Sup35 readily forms liquid assemblies, as well as gels and fibers.

[0060] 2. IUPred2 Analysis:

[0061] The full-length sequence of the Sup35 prion protein (NP_010457.3) from *Saccharomyces cerevisiae* was predicted using the IUPred2 platform. The IUPred2 analysis results are as follows: Figure 2 As shown, the results indicate that the first half of Sup35 (approximately positions 1-240, including the N / M region) is generally "loose and disordered," and ANCHOR2 (potential binding sites in the disordered region) shows that this region has many "binding hotspots," indicating that this part is very good at sticking to other molecules (proteins, RNA) and easily forms droplets; the second half (C-terminus) is mostly "ordered and structured" functional regions, which are not easy to form droplets on their own, but there are several small flexible loops at approximately 540-620, which may be involved in regulation or conditional binding.

[0062] 3. PLAAC Analysis:

[0063] The full-length sequence of Saccharomyces cerevisiae Sup35 prion protein (NP_010457.3) was predicted using the PLAAC platform. The PLAAC analysis results are as follows: Figure 3 As shown, PLAAC analysis reveals that 1-133AA is a PrD. Like prion domain.

[0064] Comprehensive analysis using FuzDrop, IUPred2, and PLAAC indicates that the N-terminal 1-240AA region of the Saccharomyces cerevisiae Sup35 prion protein is a candidate region with very strong potential to drive phase separation through multivalent interactions.

[0065] Example 2: Investigation of the gel-forming properties of self-assembled peptides of different lengths

[0066] 1. Test method:

[0067] Based on the biological prediction analysis results of Example 1, three self-assembled peptide segments of different lengths, namely 1-61AA (SEQ ID NO.2), 1-200AA (SEQ ID NO.3), and 1-240AA (SEQ ID NO.1), were selected from 1-240AA for comparative analysis of their gelation properties.

[0068] (1) Preparation of self-assembled peptides of different lengths:

[0069] The coding gene sequences of 1-61AA (SEQ ID NO.2), 1-200AA (SEQ ID NO.3), and 1-240AA (SEQ ID NO.1) were ligated into the pET-28a(+) vector to construct recombinant expression vectors, which were then transformed into BL21(DE3) to obtain recombinant bacteria BL21(DE3)-pET28a-Sup35(1-240AA), BL21(DE3)-pET28a-Sup35(1-61AA), and BL21(DE3)-Sup35(1-200AA).

[0070] The recombinant bacteria were inoculated into Erlenmeyer flasks containing 50 μg / mL kanamycin in LB liquid medium and cultured overnight at 37°C and 180 rpm with a shaking incubator for 16-18 hours. The following day, using the overnight culture as seed culture, the bacteria were inoculated into Erlenmeyer flasks containing 50 μg / mL kanamycin in a 1:100 volume ratio and cultured at 37°C and 180 rpm with a shaking incubator for 2-3 hours. OD 600nm When the concentration reached 0.3-0.6, expression was induced. The final IPTG concentration was 0.5 mM, and induction lasted 4 h. The bacterial culture was then collected and stored, and a portion was centrifuged. The centrifuged bacterial sludge was resuspended in an equal volume of PBS. The resuspended bacterial culture was then sonicated on ice at 200 W for 2 seconds, followed by a 3-second pause, for a total of 99 sonications. After sonication, the culture was centrifuged at 12000 rpm for 5 min to separate the supernatant and precipitate. The precipitate was dissolved in an equal volume of PBS. 20 μL of the unsonicated bacterial culture, the sonicated supernatant, and the PBS-dissolved sonicated precipitate were each placed in centrifuge tubes. 5 μL of 5×SDS loading buffer was added to each tube and mixed thoroughly. All samples were then boiled in a water bath for 10 min, centrifuged at 12000 rpm for 1 min, and the supernatant was collected for SDS-PAGE electrophoresis.

[0071] The supernatant after sonication was purified using an Incyte protein purification system via Ni affinity chromatography. The supernatant was thoroughly mixed with Binding Buffer (20 mM Tris-HCl, 10 mM imidazole, 0.5 M NaCl) at a 1:1 ratio and passed through a 0.22 μm bacterial filter. The filtered liquid was then passed through a pre-equilibrated Ni affinity chromatography column at a flow rate of 10 column volumes. After complete column passage, the column was washed with 15 column volumes of Binding Buffer to remove contaminating proteins. The target protein was then eluted with Elution Buffer (20 mM Tris-HCl, 500 mM imidazole, 0.5 M NaCl), and the elution peak was collected. The collected eluted protein was desalted using a G25 desalting column with a replacement buffer of 20 mM Tris-HCl.

[0072] The desalted target protein was freeze-dried to prepare freeze-dried powders of sup35(1-61AA), sup35(1-200AA), and sup35(1-240AA).

[0073] (2) Determination of gelling properties:

[0074] Sup35 (1-61AA), Sup35 (1-200AA), and Sup35 (1-240AA) lyophilized powders were dissolved in pure water at a concentration of 1% (mass fraction). If the sample did not flow at all, maintained its shape, and no visible droplets fell, it was determined that a self-supporting hydrogel had formed; the time at which this occurred was recorded as the gelation time.

[0075] 2. Test Results:

[0076] The results showed that BL21(DE3)-pET28a-Sup35(1-240AA), BL21(DE3)-Sup35(1-200AA), and BL21(DE3)-pET28a-Sup35(1-61AA) were significantly expressed at 35kDa, less than 35kDa, and less than 10kDa, respectively, which was consistent with the expected size. The expression level of BL21(DE3)-Sup35(1-200AA) was higher than that of BL21(DE3)-pET28a-Sup35(1-240AA) and BL21(DE3)-pET28a-Sup35(1-61AA).

[0077] The gelation performance test results showed that the gelation time of Sup35 (1-61AA) was about 8 hours, the gelation time of Sup35 (1-200AA) was 5 minutes, and the gelation time of Sup35 (1-240AA) was about 1 hour.

[0078] Considering both expression level and gelation properties, Sup35(1-200AA) was selected as the self-assembling peptide for subsequent experiments.

[0079] Example 3: Preparation of self-assembled superelastic protein

[0080] The self-assembled peptide SUP35 (1-200AA) shown in SEQ ID NO.3 of Example 2, the human elastin shown in SEQ ID NO.4, and the human adhesion peptide (VAPG) are combined to form the self-assembled superelastic protein shown in SEQ ID NO.6, named SUPELN. The preparation method is as follows:

[0081] 1. Construction of recombinant plasmids expressing self-assembled superelastic protein:

[0082] The SUPELN encoding gene (SEQ ID NO.5) was ligated into the pET-28a(+) vector to construct a recombinant plasmid expressing self-assembled superelastic protein. Figure 4 ).

[0083] 2. Construction of recombinant bacteria expressing self-assembled superelastic protein:

[0084] (1) Preparation of competent Escherichia coli cells:

[0085] A single colony of *Escherichia coli* BL21(DE3) was picked and inoculated into 5 mL of LB broth and incubated overnight at 37°C and 180 rpm in a shaker for 15 h. The overnight culture was then added to fresh LB broth at a 1:100 (v / v) ratio and incubated at 37°C and 180 rpm in a shaker until OD (dose elapsed). 600nm Accumulate to 0.3-0.6 (within 3 hours). Add the bacterial culture to a pre-chilled 50 mL centrifuge tube and cool on ice for 10 min. Centrifuge at 4°C and 3000 rpm for 10 min, discard the supernatant, collect the bacterial cells, resuspend the cells in pre-chilled 0.1 mol / L CaCl2 solution, and incubate on ice for 20 min. Centrifuge again at 4°C and 3000 rpm for 10 min, discard the supernatant, resuspend in 0.1 mol / L CaCl2 solution containing 15% glycerol, mix well, and aliquot into 1.5 mL centrifuge tubes, 100 μL per tube. Store at -80°C for later use to prepare competent Escherichia coli cells.

[0086] (2) Transformation:

[0087] Add 1 μL of the recombinant plasmid prepared in step 1 to 100 μL of E. coli competent cells, incubate on ice for 30 min, then heat shock in a 42℃ water bath for 90 s, and incubate on ice for another 3-5 min. Add 1 mL of fresh LB liquid medium to the treated competent cells and incubate on a shaker at 37℃ and 180 rpm for 1 h. After centrifugation, discard 400 μL of the supernatant, gently mix the remaining medium with a pipette tip, and spread 200 μL onto a plate containing 50 μg / mL kanamycin on an LB solid medium plate. Incubate at 37℃ for approximately 18 h.

[0088] (3) Screening for positive clones:

[0089] Colonies from the above-mentioned LB solid medium plates containing kanamycin were inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 180 rpm for about 15 h. PCR identification was performed using the cultured bacterial solution as a template. PCR-positive bacterial solutions were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Recombinant bacteria expressing self-assembled superelastic protein were identified by correct sequencing results.

[0090] 3. Protein expression and identification:

[0091] The recombinant bacteria expressing self-assembled superelastic protein constructed in step 2 were inoculated into LB liquid medium at a 1% inoculum and cultured overnight at 37°C and 180 rpm for 15 h using a constant temperature shaker. The overnight culture was then added to fresh LB liquid medium at a 1:100 ratio and cultured at 37°C and 180 rpm until OD (outlet capacity) was reached. 600nm To a final concentration of 0.5 mM IPTG, add to a volume of 0.3-0.6 within 3 hours and incubate at 37°C and 180 rpm for 4 hours. Centrifuge at 4°C and 5000 rpm for 10 minutes, discard the supernatant, and mix the bacterial cells with an equal volume of PBS. Sonicate the cells in an ice bath: 200 W, 2 seconds sonication followed by a 3-second pause, for a total of 99 sonications. Centrifuge and collect the supernatant and precipitate. Mix the precipitate with an equal volume of PBS. Take 20 μL each of the fermentation broth, the sonicated supernatant, and the PBS-dissolved sonicated precipitate into centrifuge tubes. Add 5 μL of 5×SDS loading buffer to each tube and mix well. Incubate all samples in a boiling water bath for 10 minutes, centrifuge at 12000 rpm for 1 minute, and collect the supernatant for SDS-PAGE electrophoresis. Collect all supernatants for protein purification.

[0092] The supernatant after sonication was purified using an Incyte protein purification system via NiN affinity chromatography. The supernatant was thoroughly mixed with Binding Buffer (20 mM Tris-HCl, 10 mM imidazole, 0.5 M NaCl) at a 1:1 ratio and passed through a 0.22 μm bacterial filter. The filtered liquid was then passed through a pre-equilibrated NiN affinity chromatography column at a flow rate of 10 column volumes. After complete column passage, the column was washed with 15 column volumes of Binding Buffer to remove contaminating proteins. The target protein was then eluted with Elution Buffer (20 mM Tris-HCl, 500 mM imidazole, 0.5 M NaCl), and the elution peak was collected. The collected eluted protein was desalted using a G25 desalting column with a replacement buffer of 20 mM Tris-HCl. The purified and desalted protein was analyzed by SDS-PAGE.

[0093] SDS-PAGE test results are as follows: Figure 5 As shown, Figure 5 The results from the A-test show that a significant target band (approximately 35.5 kDa) appears in the fermentation broth between 35 kDa and 45 kDa, and the target protein is mainly present in the supernatant after ultrasonic disruption. Figure 5 B in the figure represents the purified self-assembled superelastic protein SUPELN.

[0094] The purified and desalted target protein was freeze-dried to prepare freeze-dried powder of self-assembled superelastic protein SUPELN.

[0095] Example 4: Preparation of hydrogel

[0096] The protein concentration in the lyophilized powder of the self-assembled superelastic protein SUPELN prepared in Example 3 was determined using the BCA protein concentration assay kit.

[0097] The lyophilized powder of the self-assembled hyperelastic protein obtained in Example 3 was dissolved in pure water to obtain protein solutions of different concentrations. These protein solutions were placed in EP tubes and allowed to stand at room temperature. The EP tubes containing the protein solutions were then inverted. If the sample did not flow at all, maintained its shape, and no visible droplets fell, it was determined that a self-supporting hydrogel had formed. The time at this point was recorded as the gelation time. The gelled self-assembled hyperelastic protein in the EP tubes was sent to Beijing Zhongke Baize Technology Service Co., Ltd. for low-voltage transmission electron microscopy to observe the nanostructure of the self-assembled hyperelastic protein.

[0098] The results showed that the self-assembled superelastic protein SUPELN could gel at a concentration of 0.7 mg / mL or higher; at a concentration of 0.7 mg / mL, the gelation time was 10 min.

[0099] The hydrogel formed by allowing a 2 mg / mL aqueous solution of self-assembled superelastic protein (SUPELN) to stand at room temperature was observed using low-voltage transmission electron microscopy. The results are as follows: Figure 6 As shown, the results indicate that the hydrogel formed by the self-assembled superelastic protein SUPELN is in a nanofiber state at the microscopic level, with long fibers arranged randomly.

[0100] Comparative Example 1:

[0101] Following the method of Example 3, the self-assembled peptide SUP35 (1-200AA) shown in SEQ ID NO.3 and the human elastin shown in SEQ ID NO.4 were combined to form a self-assembled elastin, the amino acid sequence of which is shown in SEQ ID NO.7.

[0102] Comparative Example 2:

[0103] Following the method of Example 3, the self-assembling peptide SUP35 (1-200AA) shown in SEQ ID NO.3 and the human adhesion peptide (VAPG) were combined to form a self-assembling elastin, the amino acid sequence of which is shown in SEQ ID NO.8.

[0104] Experimental Example 1: Performance Evaluation of Self-Assembled Hyperelastic Protein

[0105] (1) High temperature resistance test:

[0106] The lyophilized powder of the self-assembled superelastic protein obtained in Example 3 was dissolved in pure water, placed in a 1.5 mL centrifuge tube, and the accurate protein concentration was determined using the BCA method to obtain a protein solution with a concentration of 10 mg / mL.

[0107] The protein solution was treated at room temperature (control), 50°C, 60°C, 70°C, 80°C, 85°C, 90°C, and 100°C for 1 hour, respectively. After returning to room temperature, the solution was allowed to stand and the gelation process was observed. The gelation was determined according to the method in Example 4.

[0108] The results are as follows Figure 7 As shown, the results indicate that the self-assembled hyperelastic protein prepared in this invention can form hydrogels after treatment at temperatures ranging from room temperature to 100°C. This demonstrates that the self-assembled hyperelastic protein prepared in this invention has high-temperature resistance and retains its gel-forming properties after high-temperature treatment.

[0109] (2) Investigation of rheological properties:

[0110] The rheological properties of the self-assembled superelastic protein of Example 3 and the self-assembled elastin prepared in Comparative Examples 1 and 2 were identified using a strain-controlled rheometer (TA Instruments, AR-G2); the self-assembled peptide SUP35 (1-200AA) shown in SEQ ID NO.3 was used as a control.

[0111] The storage modulus (G'), loss modulus (G''), and critical strain (γc) were measured respectively, and the G' / G'' value was calculated.

[0112] The results are shown in Table 1.

[0113] Table 1: Results of Rheological Property Investigation

[0114]

[0115] G' / G'': When the ratio is >1, elasticity dominates, and the system is a gel / elastomer; when the ratio is <1, viscosity dominates, and the system is a sol / dilute solution; when the ratio = 1, it is the critical gel point for the sol-gel transition, which is the core threshold for judging whether hyperelastic protein has completed effective self-assembly. The higher the G' / G'' ratio, the stronger the self-assembly ability, the better the hyperelastic properties, and the denser and more stable the network.

[0116] Critical strain (γc): characterizes the deformation / shear stability of the self-assembled network of hyperelastic protein; the higher the γc, the greater the deformation / shear of the protein three-dimensional network without being destroyed, the stronger the structural stability and mechanical toughness, and the more complete the elastic network can be maintained under the action of external forces (such as tissue movement in vivo and material processing shear), and the more significant the hyperelastic properties.

[0117] The results showed that the self-assembling peptide SUP35 (1-200AA), human elastin, and human adhesion peptide (VAPG) selected in this invention to form a self-assembling hyperelastic protein have a significant synergistic effect on improving the G' / G'' and γc of the self-assembling hyperelastic protein.

[0118] Experimental Example 2: Effect of self-assembled superelastic protein on the proliferation of BALB / c 3T3 cells

[0119] 1. Test method:

[0120] (1) Cell resuscitation: BALB / c 3T3 cells were taken out from the liquid nitrogen tank and rapidly revived at 37°C. The revived cells were added to centrifuge tubes containing DMEM high glucose medium, centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cell pellet was resuspended in DMEM high glucose medium containing 10% FBS, then transferred to cell culture flasks and cultured in a CO2 incubator at 37°C.

[0121] (2) Cell passage: When the cells in the cell culture flask reach a confluence of over 90%, passage is performed. Discard the culture medium in the flask, wash with PBS 1-3 times, add trypsin for digestion, and when all cells become round, discard the trypsin, add DMEM high-glucose medium containing 10% FBS, and use a pipette tip to aspirate the culture medium to wash the cells off the wall of the cell culture flask. After washing, mix the cells and passage them at a ratio of 1:3, dispensing them into 3 cell culture flasks. The passaged cells are then placed in a 37°C CO2 incubator for culture.

[0122] (3) Cell plating: After passaged cells reach a confluence of over 90%, discard the culture medium, wash three times with PBS, and then digest with trypsin. Once all cells have become rounded, pipette the cells with DMEM high-glucose medium containing 10% FBS to completely detach the cells and mix them thoroughly. Count the cells using a cell counting chamber. Seed 100 μL of the cells at a density of 2000 cells / well in 96-well cell culture plates and incubate at 37°C in a CO2 incubator for 24 h. After the cells have completely adhered, discard the supernatant. In the control group, no cells were added to the wells, only 100 μL of DMEM high-glucose medium containing 10% FBS was added.

[0123] (4) Cell proliferation experiment: The self-assembled superelastic protein lyophilized powder prepared in Example 3 was prepared into protein solutions of different concentrations (0.25, 0.125, 0.0625, 0.03125 mg / mL) using PBS, and then filtered and sterilized using a bacterial filter. Commercially available collagen was processed in the same way and also prepared into protein solutions of different concentrations (0.25, 0.125, 0.0625, 0.03125 mg / mL). The above protein solutions of different concentrations were added to the 96-well culture plates containing cells in step (3), 100 μL per well, with 8 replicates for each concentration, and 30 μL of DMEM high-glucose medium containing 2% FBS was added to each well. A control group and a blank group were set up, with 8 replicates in each group. In the control group, 100 μL of PBS + 30 μL of DMEM high-glucose medium containing 2% FBS was added to each well. In the blank group, each well was replaced with 100 μL of PBS + 30 μL of DMEM high-glucose medium containing 2% FBS. The mixtures were incubated at 37°C in a CO2 incubator for 24 h.

[0124] (5) Cell proliferation was determined using a CCK-8 assay kit. The specific method is as follows: Remove the old culture medium from the wells of the cell culture plate from step (4), add 100 μL of DMEM high-glucose medium containing 10% CCK-8 to each well, and incubate in the dark for 4 h. After incubation, measure the absorbance at 450 nm. Calculate the cell proliferation rate based on the average absorbance of each group. The formula for calculating the cell proliferation rate is: Cell proliferation rate % = (As-Ab) / (Ac-Ab) × 100%

[0125] In the formula: As is the absorbance of the sample group, Ab is the absorbance of the blank group, and Ac is the absorbance of the control group.

[0126] 2. Test Results:

[0127] Table 2: Effects of self-assembled superelastic protein on the proliferation of BALB / c 3T3 cells

[0128]

[0129] Note: Different lowercase letters in the same row of shoulder labels indicate significant differences (P < 0.05), while the same or no shoulder labels indicate no significant differences (P > 0.05).

[0130] The results are shown in Table 2. The self-assembled hyperelastic protein significantly enhanced the proliferation of BALB / c 3T3 cells compared to the control group. Concentrations of 0.25, 0.125, 0.0625, and 0.03125 mg / mL of self-assembled hyperelastic protein increased the proliferation of BALB / c 3T3 cells by 121.00%, 55.20%, 39.66%, and 33.16%, respectively, compared to the control group. Furthermore, the self-assembled hyperelastic protein exhibited superior proliferation effects on BALB / c 3T3 cells compared to commercially available collagen, increasing the proliferation by 60.48%, 25.30%, 22.11%, and 18.18%, respectively.

[0131] Experimental Example 3: Effect of self-assembled superelastic protein on the migration of BALB / c 3T3 cells

[0132] 1. Test method:

[0133] (1) Cell scratch assay was performed using 6-well cell culture plates. BALB / c3T3 cells in logarithmic growth phase cultured in cell flasks were digested with trypsin, then resuspended in DMEM high-glucose medium containing 10% FBS and mixed thoroughly. The cells were then incubated at approximately 3 × 10⁻⁶ wells. 5 Inoculate 2 mL of cells per well into 6-well cell culture plates. The density should be such that the cell confluence exceeds 90% when the scratching begins. Make sure to mix the cells thoroughly during inoculation and continue culturing until the cells adhere to the plate.

[0134] (2) Remove the culture medium, use a 200μL pipette tip and a ruler to make a "#" shaped scratch. Use the same pipette tip to make the scratches as consistent as possible between different wells. Wash with PBS 3 times to remove free cells and cell debris caused by the scratches. Observe that the scratches are clear, straight and without cell residue.

[0135] (3) Calculation of cell migration rate: The self-assembled superelastic protein lyophilized powder and commercially available collagen prepared in Example 3 were diluted with DMEM high-glucose medium containing 4% FBS to obtain protein concentrations of 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, and 0.03125 mg / mL, respectively. 2 mL of each solution was added to the wells of the cell culture plates after the above treatment, with each treatment repeated in triplicate. Three wells were also set up as a blank control group. 2 mL of DMEM high-glucose medium containing 4% FBS was added to each well of the blank control group. Photos were taken at 0 and 24 h. The scratch area of ​​each group was calculated using ImageJ software, and the cell migration rate was calculated using the following formula:

[0136] Cell scratch migration rate (%) = (Initial scratch area - Final scratch area) / Initial scratch area × 100%

[0137] 2. Test Results:

[0138] Photos taken at 0 and 24 hours after cell scratch treatment are shown below. Figure 8 As shown; cell scratch migration rate results are as follows. Figure 9 As shown in the figure, the migration rate of BALB / c 3T3 cells in each of the different concentrations of self-assembled hyperelastic protein sample groups was significantly higher than that in the control group. At concentrations of 0.25, 0.125, 0.0625, and 0.03125 mg / mL, the cell migration rate of the self-assembled hyperelastic protein sample groups was increased by 148%, 145%, 127%, and 113% compared with the control group, respectively, and by 58.59%, 29.63%, 13.09%, and 9.86% compared with the commercially available collagen group, respectively. This indicates that self-assembled hyperelastic protein is beneficial in promoting the migration of BALB / c 3T3 cells.

[0139] Experimental Example 4: Effect of self-assembled superelastic protein on BALB / c 3T3 cell adhesion

[0140] 1. Test method:

[0141] (1) Coating culture plates: The self-assembled superelastic protein lyophilized powder prepared in Example 3 and commercially available collagen were diluted with PBS to prepare protein solutions with concentrations of 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL and 0.03125 mg / mL, respectively, as the sample group; the control group was 1% BSA (bovine serum albumin); 100 μL was added to each well of a 96-well cell culture plate, and each treatment was repeated in 8 wells, and incubated overnight at 4°C.

[0142] (2) BSA blocking: After overnight incubation at 4°C, wash 3 times with PBS; block with 1% BSA at 37°C for 1 h, and rinse 3 times with PBS to block the uncoated areas.

[0143] (3) Cell preparation: When the BALB / c 3T3 cells in the cell culture flask reach more than 90%, discard the culture medium in the flask, wash with PBS 1-3 times, add trypsin for digestion, and when all the cells become round, discard the trypsin, add DMEM high glucose culture medium containing 4% FBS, use the pipette tip to draw the culture medium to wash the cells off the wall of the cell culture flask, mix the washed cells, and use a cell counting chamber to count the cells.

[0144] (4) Cell seeding: BALB / c 3T3 cells were seeded at 3500 cells / well in a 96-well plate after step (2). A blank control group was set up with culture medium but no cells, and 8 replicate wells were set up in each group.

[0145] (5) Cell culture: Incubate at 37°C for 24 hours so that cells can adhere to proteins. Wash away unadhered cells with PBS and keep only the adhered cells.

[0146] (6) Cell count detection: The CCK-8 assay kit was used to quantitatively detect the adherent cells. The detection wavelength was 450 nm, and the relative cell adhesion rate was calculated based on the average absorbance of each group.

[0147] Relative cell adhesion rate = (As-Ab) / (Ac-Ab) × 100%

[0148] In the formula: As is the absorbance of the sample group, Ab is the absorbance of the blank group, and Ac is the absorbance of the control group.

[0149] 3. Test Results:

[0150] The results are as follows Figure 10 As shown in the figure. The results showed that the self-assembled hyperelastic protein at concentrations of 0.25, 0.125, 0.0625, and 0.03125 mg / mL significantly enhanced the adhesion of BALB / c 3T3 cells to the control group, increasing by 27.85%, 23.74%, 19.43%, and 16.54% respectively, indicating that the self-assembled hyperelastic protein can effectively promote cell adhesion.

[0151] Experimental Example 5: Effect of self-assembled superelastic protein on UVB irradiation damage in BALB / c 3T3 cells

[0152] 1. Test method:

[0153] 1.1 Injury Prevention Experiment:

[0154] BALB / c 3T3 cells were seeded at a density of 3000 cells / well in 96-well plates and cultured adherently for 24 h. The self-assembled superelastic protein prepared in Example 3 and commercially available collagen were diluted with DMEM high-glucose medium containing 4% FBS to obtain concentrations of 0.25, 0.125, 0.0625, and 0.03125 mg / mL, respectively, serving as sample groups. The experiment included sample groups, a blank control group, and a model group. 100 μL of DMEM high-glucose medium containing 10% FBS was added to the blank control group and the model group, while 100 μL of protein solution of different concentrations was added to each well of the sample group. Each treatment was repeated in 8 wells. The plates were incubated in a CO2 incubator for 24 h. The supernatant was aspirated, and the cells were washed twice with PBS. 100 μL of PBS was added to each well of the 96-well plate. The sample and model groups were irradiated under UV light for 3 min, while the blank control group was not irradiated. Cell viability was measured using a CCK-8 assay kit. Observe the cell survival status of each group, and determine the OD of each group. 450nm The average value is used to calculate cell viability.

[0155] Cell viability = (OD of treatment group) 450nm / Blank control group OD 450nm ) × 100%

[0156] The treatment group refers to either the sample group or the model group.

[0157] 1.2 Intervention injury experiment:

[0158] BALB / c 3T3 cells were seeded at a density of 3000 cells / well in 96-well plates and cultured adherently for 24 h. After washing once with PBS, 100 μL of PBS was added to each well of the 96-well plates. The sample and model groups were irradiated under UV light for 8 min, while the blank control group was not irradiated with UV light. The PBS was removed from each well. The sample groups were treated with self-assembled superelastic protein and commercially available collagen diluted in DMEM high-glucose medium containing 4% FBS, with protein concentrations of 0.25, 0.125, 0.0625, and 0.03125 mg / mL, respectively. The blank control and model groups were treated with only DMEM high-glucose medium containing 4% FBS, 100 μL per well. Each treatment was repeated in 8 wells. The cells were cultured in a CO2 incubator for 24 h, and cell viability was determined using a CCK-8 assay kit. Cell viability was observed in each group, and the OD values ​​were calculated based on the cell growth rate. 450nm The average value is used to calculate cell viability.

[0159] Cell viability = (OD of treatment group) 450nm / Blank control group OD 450nm ) × 100%

[0160] 2. Test Results:

[0161] 2.1 Results of the injury prevention experiment:

[0162] The experimental results for injury prevention are shown in Figure 11 Compared with the blank control group without UVB irradiation, the cell survival rate of the model group irradiated with UVB was significantly decreased. After UVB irradiation, the cell survival rate of the model group without protein treatment was 86.55%; the cell survival rate of the group supplemented with self-assembled hyperelastic protein at concentrations of 0.03125-0.25 mg / mL reached 89.93%-94.63%, significantly higher than that of the model group. This indicates that self-assembled hyperelastic protein has a certain preventive effect against UV damage.

[0163] 2.2 Results of the intervention injury experiment:

[0164] Experimental results of intervention for injury are shown in Figure 12In the intervention group, the cell survival rate of the model group without protein treatment was 68.15%; the cell survival rate of the group supplemented with self-assembled hyperelastic protein at concentrations of 0.03125-0.25 mg / mL ranged from 73.52% to 78.71%, which was 7.88%-15.49% higher than that of the model group, and significantly higher. This indicates that self-assembled hyperelastic protein has a certain repair effect on oxidative damage to cells.

[0165] Experimental Example 6: Determination of DPPH scavenging capacity of self-assembled superelastic protein

[0166] 1. Test method:

[0167] The self-assembled hyperelastic protein lyophilized powder prepared in Example 3 was diluted with PBS to prepare protein solutions with concentrations of 0.5, 1.0, 3.0, 5.0, and 10.0 mg / mL. The sample group consisted of a mixture of 1 mL of protein solutions of different concentrations (0.5, 1.0, 3.0, 5.0, and 10.0 mg / mL) and 1 mL of 0.1 mmol / L DPPH ethanol solution; the control group consisted of a mixture of 1 mL of 0.1 mmol / L DPPH ethanol solution and 1 mL of anhydrous ethanol; and the blank group consisted of a mixture of 1 mL of self-assembled hyperelastic protein solutions of different concentrations and 1 mL of anhydrous ethanol. All mixtures were reacted at room temperature in the dark for 30 min, then centrifuged at 8000 r / min for 1 min. The supernatant was measured at 517 nm. The experiment was independently repeated three times. The DPPH scavenging rate was calculated based on the absorbance. The formula for calculating the DPPH scavenging rate is as follows:

[0168] DPPH clearance rate (%) = [Ac - (As - Ab)] / Ac × 100%

[0169] In the formula: As, Ac, and Ab are the average absorbance values ​​of the sample group, control group, and blank group, respectively.

[0170] 2. Test Results:

[0171] The results are shown in Table 3:

[0172] Table 3: Results of DPPH scavenging ability assay of self-assembled superelastic protein

[0173]

[0174] Antioxidant active substances can reduce the number of DPPH free radicals by pairing with their unpaired electrons, resulting in a lighter solution color. The intensity of the color indicates the substance's ability to scavenge DPPH free radicals. The results showed that the DPPH scavenging rates of self-assembled hyperelastic protein at concentrations of 10.0, 5.0, 3.0, 1.0, and 0.5 mg / mL were 65.32%, 53.44%, 46.84%, 34.35%, and 23.83%, respectively. These results indicate that self-assembled hyperelastic protein can reduce the oxidative damage caused by DPPH in the body.

[0175] Experimental Example 7: Determination of ·OH scavenging ability of self-assembled superelastic protein

[0176] 1. Test method:

[0177] The self-assembled hyperelastic protein lyophilized powder prepared in Example 3 was diluted with PBS to prepare protein solutions with concentrations of 0.5, 1.0, 3.0, 5.0, and 10.0 mg / mL. The sample group consisted of 500 μL of 9.0 mM FeSO4·7H2O solution, 500 μL of 9.0 mM salicylic acid solution (dissolved in ethanol), 500 μL of protein solutions at each concentration (0.5, 1.0, 3.0, 5.0, and 10.0 mg / mL), and 500 μL of 8.8 mM H2O2 added sequentially to a 2 mL centrifuge tube. The control group consisted of replacing the hyperelastic protein solution in the sample group with an equal volume of distilled water, while keeping other components and their volumes unchanged. The blank group consisted of replacing the H2O2 in the sample group with an equal volume of deionized water, while keeping other components and their volumes unchanged. After mixing, the solutions were heated in a water bath at 37°C for 30 min. After centrifugation at 8000 r / min for 1 min, the supernatant was collected and its absorbance was measured at 510 nm. The experiment was independently repeated three times. The ·OH scavenging rate was calculated based on the absorbance. The formula for calculating the ·OH scavenging rate is as follows:

[0178] OH removal rate (%) = [Ac-(As-Ab)] / Ac × 100%

[0179] In the formula: As, Ac, and Ab are the average absorbance values ​​of the sample group, control group, and blank group, respectively.

[0180] 2. Test Results:

[0181] The results are shown in Table 4. Figure 13 As shown:

[0182] Table 4: Results of the determination of the ·OH scavenging ability of self-assembled superelastic protein

[0183]

[0184] •OH can react with some functionally active biomolecules in the human body, causing them to lose their original function and thus resulting in damage to the body. The scavenging rates of •OH by hyperelastic protein at concentrations of 10.0, 5.0, 3.0, 1.0, and 0.5 mg / mL were 84.66%, 76.16%, 52.00%, 16.76%, and 9.35%, respectively. The results indicate that the scavenging rate of •OH by self-assembled hyperelastic protein is positively correlated with its concentration; higher concentrations significantly reduce •OH scavenging and thus minimize damage to the body.

[0185] Experimental Example 8: Determination of ABTS scavenging ability of self-assembled superelastic protein

[0186] 1. Test method:

[0187] Preparation of ABTS test solution:

[0188] ABTS stock solution (7.4 mmol / L, 0.4 mL): Take 0.0045 g of ABTS and add 1.1025 mL of distilled water (MW=548.7); K2S2O8 stock solution (2.6 mmol / L, 1.43 mL): Take 0.0025 g of K2S2O8 and add 3.575 mL of distilled water (MW=270.32). Prepare a mixture by mixing 1 mL of 7.4 mM ABTS with 1 mL of 2.6 mM K2S2O8 and incubate in the dark at room temperature for 15 h. Dilute the mixture 50 times with PBS (pH 7.4) to obtain the ABTS working solution. The sample group consisted of a mixture of 400 μL of self-assembled hyperelastic protein solution at different concentrations (0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 1.0, 3.0, 5.0, 10.0 mg / mL) and 1.6 mL of ABTS working solution. The control group consisted of a mixture of 400 μL of deionized water and 1.6 mL of ABTS working solution. The blank group consisted of a mixture of 400 μL of self-assembled hyperelastic protein solution at different concentrations (0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 1.0, 3.0, 5.0, 10.0 mg / mL) and 1.6 mL of deionized water. After standing for 6 min, the mixture was centrifuged at 8000 r / min for 1 min. The supernatant was then measured at 734 nm. The experiment was independently repeated three times. The formula for calculating ABTS clearance rate is as follows:

[0189] ABTS radical scavenging rate (%) = [Ac - (As - Ab)] / Ac × 100%

[0190] In the formula: As, Ac, and Ab are the average absorbance values ​​of the sample group, control group, and blank group, respectively.

[0191] 2. Test Results:

[0192] The results are shown in Table 5. Figure 14 As shown, when the concentrations of self-assembled superelastic protein were 0.5, 1.0, 3.0, 5.0, and 10.0 mg / mL, the ABTS free radical scavenging rate could reach 100%. In the range of 0.0025 mg / mL to 0.3 mg / mL, the ABTS free radical scavenging rate was positively correlated with the concentration, and the scavenging ability increased with increasing concentration.

[0193] Table 5: Results of ABTS scavenging ability assay of self-assembled superelastic protein

[0194]

[0195] ABTS (2,2'-adiazono-3-ethylbenzothiazoline-6-sulfonic acid) generates green ABTS radical cations under the action of oxidants. In the presence of antioxidants, these cations react with the ABTS radical cations, causing the color to lighten. The ABTS radical scavenging assay is used as a standard tool for assessing in vitro antioxidant capacity. The results show that self-assembled hyperelastin concentrations of 0.0025-0.5 mg / mL achieve ABTS scavenging rates of 15.84-100%, and concentrations of 0.5 mg / mL and above demonstrate a strong 100% ABTS scavenging capacity. This indicates that self-assembled hyperelastin possesses superior antioxidant capacity, and that concentration and scavenging rate are positively correlated.

[0196] Test Example 9: Weight Adhesion Test

[0197] 1. Test method:

[0198] Weigh 10 mg of the self-assembled superelastic protein freeze-dried powder prepared in Example 3 and dissolve it in 4 µL of water on the surface of 500 g and 100 g weights respectively. Adhere the weights to the surface using a weight box, let stand for 3 min, and observe the adhesion effect.

[0199] Weigh 10 mg of the self-assembled superelastic protein freeze-dried powder, add 4 µL of water and dissolve it on the surface of a 100 g weight. Adhere the mixture with a 10 μL white pipette tip, let it stand for 3 min, and observe the adhesion effect.

[0200] 2. Test Results:

[0201] The results are as follows Figure 15 As shown, the self-assembled hyperelastic protein can adhere to 100g and 500g weights using a weight box; the self-assembled hyperelastic protein can adhere to 100g weight using a 10μL white pipette tip. The results indicate that the self-assembled hyperelastic protein prepared in this invention has excellent adhesion properties.

[0202] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A self-assembled superelastic protein, characterized in that, include: Self-assembling peptide SUP35, human elastin and human adhesion peptide; The amino acid sequence of the self-assembled peptide SUP35 is shown in SEQ ID NO.3; the amino acid sequence of the human elastin is shown in SEQ ID NO.4; and the sequence of the human adhesion peptide is VAPG. The amino acid sequence of the self-assembled superelastic protein is shown in SEQ ID NO.

6.

2. The self-assembled superelastic protein according to claim 1, characterized in that, The self-assembled superelastic protein is prepared by the following method: (1) Construct a recombinant plasmid containing the gene encoding self-assembled superelastic protein, and transfer the recombinant plasmid into competent Escherichia coli cells to obtain recombinant bacteria; (2) Inoculate the recombinant bacteria into LB liquid medium and culture until OD. 600nm When the concentration is 0.3-0.6, IPTG is added for induction culture; the induced culture solution is centrifuged, the bacterial cells are collected, sonicated and disrupted, centrifuged again, the supernatant is collected and purified to obtain self-assembled superelastic protein.

3. The self-assembled superelastic protein according to claim 2, characterized in that, In step (1), the nucleotide sequence of the self-assembled superelastic protein encoding gene is shown in SEQ ID NO.

5.

4. The self-assembled superelastic protein according to claim 2, characterized in that, In step (2), the final concentration of IPTG added is 0.5mM, and the induction culture conditions are: 37℃, 180rpm constant temperature shaker culture for 4h.

5. A hydrogel, characterized in that, The hydrogel is obtained by self-assembly of the self-assembled superelastic protein according to any one of claims 1-4 in a solvent; The solvent is pure water, PBS buffer, TB buffer, or cell culture medium.

6. The hydrogel according to claim 5, characterized in that, The concentration of self-assembled superelastic protein in the solvent is not less than 0.7 mg / mL.

7. The use of the self-assembled superelastic protein according to any one of claims 1-4 or the hydrogel according to claim 5 or 6 in the preparation of adhesive materials.

8. The use of the self-assembled superelastic protein according to any one of claims 1-4 or the hydrogel according to claim 5 or 6 in the preparation of culture media that promote cell proliferation, cell migration and / or cell damage repair.

9. The use of the self-assembled superelastic protein according to any one of claims 1-4 or the hydrogel according to claim 5 or 6 in the preparation of a free radical scavenger, characterized in that, The free radical is DPPH, ·OH, or ABTS.

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