A fusion protein mfp-elp with ultraviolet damage repair function and application thereof

Abstract

This invention discloses a fusion protein, Mfp-ELP, with ultraviolet (UV) damage repair function and its applications, belonging to the field of biomedical technology. The amino acid sequence of the fusion protein Mfp-ELP is shown in SEQ ID No: 1. This invention, through optimized gene design, expression system, and purification process, achieves a simple, easy-to-implement, high-purity, and low-cost preparation method with good water solubility, stability, compatibility, and biocompatibility, facilitating large-scale production. It can significantly promote the proliferation of HSF cells and UVB damage repair, accelerate the healing rate of full-thickness skin defects in mice, reduce photodamage to HSF cells, alleviate UV-induced photoaging and sunburn, and exhibit significant antioxidant activity and efficacy in reducing cell and skin sunburn. It can be widely used in drugs, medical biomaterials, and cosmetics for anti-photoaging and / or promoting skin sunburn repair and / or adjuvant treatment of photodamaged skin diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fusion protein Mfp-ELP with ultraviolet damage repair function and its application; particularly relating to the application of a recombinant fusion protein based on mussel adhesive protein (Mfp) and elastin-like polypeptide (ELP) in the prevention and treatment of skin photoaging, repair of UVB-induced skin cell damage, and adjuvant treatment of photodamaged skin diseases. Background Technology

[0002] Ultraviolet radiation, especially UVB (medium-wave ultraviolet radiation), is one of the main environmental factors causing photodamage to the skin. UVB leads to photoaging (wrinkles, sagging), inflammatory responses, and even precancerous lesions by directly damaging DNA, activating oxidative stress pathways (such as excessive ROS production), disrupting the skin barrier (loss of stratum corneum lipids, degradation of collagen fibers), and inhibiting fibroblast activity. Current repair methods have significant shortcomings.

[0003] Limitations of single-component formulations: While mussel adhesive protein possesses strong adhesion (promoting wound healing through DOPA groups) and antioxidant properties (scavenging ROS), its direct effect on collagen fiber regeneration and skin elasticity restoration is relatively weak; elastin can maintain the skin's elastic network, but its antioxidant and anti-inflammatory capabilities are insufficient, and naturally extracted elastin carries the risk of immunogenicity.

[0004] Technical bottlenecks: Chemically cross-linked mixed protein preparations are prone to problems such as active site masking and decreased biocompatibility; Although genetic engineering can achieve fusion expression, there are conflicts in the amino acid sequences of the two types of proteins (mussel adhesive protein is rich in tyrosine, and elastin contains repetitive hydrophobic sequences) in terms of folding conformation and expression efficiency, resulting in low solubility and poor functional synergy of recombinant proteins.

[0005] Currently, the application of protein fusion technology in skin repair focuses on the combination of "structural proteins + functional proteins" (such as the fusion of collagen and growth factors), but there is very little research on the synergistic effect of multiple targets such as "adhesion-antioxidation-elasticity repair".

[0006] Existing fusion approaches mostly focus on enhancing a single function (such as enhancing adhesion or prolonging half-life), for example, fusing mussel adhesive protein with hyaluronidase to promote drug penetration, but do not involve elastic repair;

[0007] The DOPA oxidative cross-linking properties of mussel adhesive proteins and the formation of hydrophobic elastic networks by elastin exhibit "conformational competition"—the former requires an oxidative environment to promote adhesion, while the latter is prone to degradation under oxidative conditions. How to balance the activities of the two through sequence design has become a key obstacle to the realization of the fusion protein's function. At the same time, there is still no experimental data to support the fusion protein's inhibitory effect on UVB-induced keratinocyte apoptosis and its regulatory mechanism on collagen synthesis in fibroblasts. Summary of the Invention

[0008] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a fusion protein, Mfp-ELP, with ultraviolet damage repair function and its applications. This invention, through optimized gene design, expression system, and purification process, solves the problems of functional limitations of single protein components and the contradiction between complex pathological mechanisms in the fields of skin photoaging and UVB damage repair, and expands its application value in medicine, cosmetics, and other fields.

[0009] The UVB damage repair market urgently needs "multifunctional synergistic" formulations. Traditional antioxidants (such as vitamin C) have poor stability, and physical sunscreens cannot repair damaged tissues. Existing medical dressings mostly focus on a single repair aspect (such as hemostasis or moisturizing). The fusion of mussel adhesive protein and elastin can theoretically achieve a closed-loop repair process of "adhesion and colonization - anti-oxidation and anti-inflammation - elastic network reconstruction".

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A fusion protein, Mfp-ELP, with UV damage repair function has the amino acid sequence shown in SEQ ID No: 1.

[0012] The gene encoding the aforementioned fusion protein Mfp-ELP.

[0013] Preferably, the nucleotide sequence of the encoding gene is shown in SEQ ID No: 2.

[0014] The aforementioned biomaterials related to the fusion protein Mfp-ELP are any one or more combinations of the following biomaterials:

[0015] (a) An expression cassette containing the above-mentioned encoded genes;

[0016] (b) Recombinant expression vectors containing the above-mentioned coding genes;

[0017] (c) A recombinant expression vector containing the expression cassette described in (a);

[0018] (d) Recombinant bacteria containing the above-mentioned encoding genes;

[0019] (e) Recombinant bacteria containing the expression cassette described in (a);

[0020] (f) Recombinant bacteria containing the recombinant expression vector described in (b) or (c).

[0021] Furthermore, the starting vector for the recombinant expression vector described in (b) and (c) is a pET series vector, etc.; preferably, it is a pET-28a(+) vector.

[0022] Furthermore, the host bacteria corresponding to the recombinant bacteria mentioned in (d), (e), and (f) are selected from prokaryotes, etc.; the prokaryotes include bacteria such as Escherichia. More specifically, the prokaryote is Escherichia coli (E. coli), specifically Escherichia coli BL21(DE3).

[0023] In one embodiment of the present invention, the coding gene sequence of the above-mentioned fusion protein Mfp-ELP is ligated to the CDS region of a vector to obtain a plasmid, and then recombinantly expressed in Escherichia coli to obtain the recombinant Mfp-ELP fusion protein. Its biocompatibility and UV repair ability are then tested. Furthermore, the above-mentioned Mfp-ELP fusion protein is prepared into a hydrogel using a PI-PAM / BACA hydrogel system. The extract of this hydrogel is subjected to tests for biocompatibility, ROS scavenging ability, and hemolytic properties, and animal experiments are conducted to investigate its ability to repair UVB damage.

[0024] The application of the encoding gene of the fusion protein Mfp-ELP or biomaterials related to the fusion protein Mfp-ELP in the preparation of the fusion protein Mfp-ELP.

[0025] The application of the aforementioned fusion protein Mfp-ELP, encoding gene, biomaterials related to the fusion protein Mfp-ELP, mussel adhesive protein (Mfp), or biomaterials related to mussel adhesive protein (Mfp) in the preparation of products resistant to UV damage.

[0026] Preferably, the application is at least one of the following:

[0027] 1) Application in the preparation of products for preventing and treating skin photoaging;

[0028] 2) Application in the preparation of products for repairing UVB-induced skin damage;

[0029] 3) Application in the preparation of products for adjuvant treatment of photodamaged skin diseases.

[0030] Preferably, the product includes at least one of the following: pharmaceuticals, medical biomaterials, and cosmetics.

[0031] The amino acid sequence of the mussel adhesive protein (Mfp) is shown as 1 to 87aa in SEQ ID No: 1;

[0032] A gene encoding the above-mentioned mussel adhesive protein (Mfp), preferably, the nucleotide sequence of the gene is shown as 1 to 261 bp in SEQ ID No: 2.

[0033] The mussel adhesive protein (Mfp)-related biomaterial is any one or a combination of the following biomaterials:

[0034] (A) An expression cassette containing the above-mentioned encoded genes;

[0035] (B) Recombinant expression vectors containing the above-mentioned coding genes;

[0036] (C) A recombinant expression vector containing the expression cassette described in (A);

[0037] (D) Recombinant bacteria containing the above-mentioned encoding genes;

[0038] (E) Recombinant bacteria containing the expression cassette described in (A);

[0039] (F) Recombinant bacteria containing the recombinant expression vector described in (B) or (C).

[0040] Furthermore, the starting vector for the recombinant expression vectors described in (B) and (C) is a pET series vector, etc.; preferably, it is a pET-28a(+) vector.

[0041] Furthermore, the host bacteria corresponding to the recombinant bacteria mentioned in (D), (E), and (F) are selected from prokaryotes, etc.; the prokaryotes include bacteria such as Escherichia. More specifically, the prokaryote is Escherichia coli (E. coli), specifically Escherichia coli BL21(DE3).

[0042] The present invention has the following advantages and effects compared with the prior art:

[0043] (1) Studies have found that, compared with monomeric proteins, the fusion protein of the present invention has significantly improved antioxidant activity, activity to reduce sunburn and oxidative damage to cells, and efficacy to reduce adverse reactions such as skin erythema, desquamation, exudation and crusting, which can greatly reduce the dosage and compatibility cost in the product.

[0044] (2) The fusion protein preparation method of the present invention is simple, easy to implement, has high purity and low cost, and has good water solubility, stability, compatibility and biocompatibility, which is convenient for large-scale production; it can significantly promote the proliferation of HSF cells and repair UVB damage, accelerate the healing rate of full-thickness skin defects in mice, reduce photodamage of HSF cells, and alleviate skin photoaging and sunburn caused by ultraviolet (UVB).

[0045] (3) The fusion protein of the present invention has significant antioxidant activity and the effect of reducing sunburn on cells and skin. It can be widely used in drugs, medical biomaterials and cosmetics that resist skin photoaging and / or promote skin sunburn repair. Attached Figure Description

[0046] Figure 1 This is a graph showing the expression results of the Mfp-ELP fusion protein; where: M: protein marker, IPTG-: bacterial culture without IPTG induction, whole liquid: bacterial culture after IPTG induction, supernatant: supernatant after cell disruption, precipitate: precipitate after cell disruption.

[0047] Figure 2 This is a diagram showing the purification results of the Mfp-ELP fusion protein.

[0048] Figure 3 This is a diagram showing the biocompatibility results of the Mfp-ELP fusion protein in HSF cells (A) and NIN / 3T3 cells (B).

[0049] Figure 4 This is a graph showing the UV repair performance of the Mfp-ELP fusion protein in HSF cells.

[0050] Figure 5 This is a diagram showing the biocompatibility results of the Mfp-ELP hydrogel extract in HSF cells.

[0051] Figure 6 This is a graph showing the results of ROS clearance in HSF cells using Mfp-ELP hydrogel extract.

[0052] Figure 7 This is a graph showing the results of a hemolysis experiment using Mfp-ELP hydrogel extract.

[0053] Figure 8 This is a picture showing the results of skin repair in mice.

[0054] Figure 9 This is an image of a HE section of mouse skin.

[0055] Figure 10 This is a picture of the Masson assay results for mouse skin.

[0056] Figure 11This is a graph showing the results of mouse skin MDA (A), SOD (B), and GPx (C) detection.

[0057] Note: ns: no significant difference, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.

[0059] Example 1

[0060] I. Experimental Materials and Reagents

[0061] Strains and plasmids: Escherichia coli strains (such as BL21(DE3)), pET-28a(+) plasmid;

[0062] Culture medium: LB solid medium and LB liquid medium, both with an appropriate amount of kanamycin added;

[0063] Protein purification reagents: nickel ion affinity chromatography column and matching buffer;

[0064] Other: PCR-related reagents, ultrasonic disruptor, etc.

[0065] II. Construction of the Expression System

[0066] Vector selection and processing: The pET-28a(+) plasmid was extracted. This plasmid carries a kanamycin resistance gene, facilitating subsequent screening. The plasmid was double-digested using NdeI and BamHI restriction endonucleases. The reaction system (total volume 50 μL as an example): 1 μg pET-28a(+) plasmid, 1 μL NdeI, 1 μL BamHI, 5 μL corresponding restriction buffer, and sterile water to a final volume of 50 μL. Incubation was performed at 37°C for 2–3 hours. The linearized plasmid was then separated and purified by agarose gel electrophoresis.

[0067] Target gene insertion: The Mfp-ELP fusion protein gene (SEQ ID No: 2) of E. coli with optimized codon length of 333 bp was obtained and added to the ligation reaction system at a molar ratio of 3:1 with the above-mentioned double-digested pET-28a(+) plasmid. Then, an appropriate amount of DNA ligase and ligation buffer were added, and the mixture was ligated at 55℃ for 30 min to construct the recombinant plasmid. The promoter on the plasmid was used to drive the subsequent efficient transcription of the Mfp-ELP fusion protein gene in the host cell.

[0068] The amino acid sequence of the Mfp-ELP fusion protein is shown in SEQ ID No: 1, where 1-87aa: Mfp, 88-92aa: linker peptide, and 93-110aa: 3 copies of ELP. The Mfp-ELP fusion protein has a histidine-like tag (see 1-15aa in SEQ ID No: 1), and the fusion protein can be purified using a nickel ion affinity chromatography column.

[0069] III. Host Cell Transformation and Screening

[0070] Transformation: The constructed recombinant plasmid was added to competent E. coli (e.g., 100 μL of pre-chilled BL21(DE3) competent cells), gently mixed, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and then quickly returned to the ice bath for 2 minutes. 900 μL of liquid medium was added, and the cells were incubated at 37°C with shaking at 220 rpm for 1 hour.

[0071] Screening: Spread an appropriate amount of the above culture onto LB solid medium containing kanamycin (final concentration 50 μg / mL) and incubate overnight at 37°C. The next day, pick a single colony and inoculate 200 μL into 1.5 mL centrifuge tubes containing kanamycin liquid medium, and incubate at 37°C with shaking at 220 rpm. Select PCR-positive clones for sequencing, and finally screen out positive clones carrying the correct recombinant plasmid.

[0072] IV. Induced Expression Regulation

[0073] Culture conditions optimization: Positive clones with correct sequencing were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid medium containing kanamycin (50 μg / mL), and cultured at 37°C with shaking at 220 rpm. OD was measured every hour. 600 When OD 600 When the growth rate reaches 0.6–0.8 (logarithmic growth phase), prepare for induction.

[0074] Inducer addition: Add IPTG to the culture system to a final concentration of 0.5–1 mM, and continue incubation at 37°C and 220 rpm for 6–7 hours with shaking to induce recombinant fusion protein gene expression. Results are as follows: Figure 1 As shown, the target protein (approximately 15 kDa) was successfully expressed in E. coli under IPTG induction, and it was mainly present in the supernatant of cell lysate as a soluble protein.

[0075] V. Protein Purification

[0076] Preliminary treatment: Collect the induced bacterial culture and centrifuge at 8000 rpm for 20 minutes at 4°C to collect bacterial cells. Resuspend the bacterial cells in Buffer A (8M Urea, 0.5M NaCl, 0.02M Tris, pH=7.0) at a ratio of 10 OD / mL, and then use a high-pressure homogenizer to disrupt the bacterial cells. Adjust the disruption pressure according to the actual situation until the bacterial solution is clear. Use an ultrasonic homogenizer to disrupt the cells. Ultrasonic conditions: 3 seconds on, 3 seconds off, total duration 10-15 minutes. Centrifuge the completely disrupted solution at 8000g for 30 minutes, collect the supernatant, and filter through a 0.22μm filter membrane to obtain the unpurified sample.

[0077] Affinity chromatography: The unpurified sample obtained above was slowly loaded onto a pre-equilibrated nickel ion affinity chromatography column at a flow rate of 1 mL / min. After loading, the column was washed with wash buffer containing 10% imidazole to remove non-specifically bound impurities.

[0078] Remove the nickel column at 4°C and allow the preservation solution to slowly drain out under gravity before adding 3 to 5 column volumes of ddH2O.

[0079] Equilibrate the nickel column with 3–5 column volumes of Buffer A (8M Urea, 0.5M NaCl, 0.02M Tris, pH=7.0). Load the filtered, unpurified sample at 5 mL intervals, allowing it to flow out under gravity; collect the eluent as flow-through. After loading, rinse the nickel column with 3–5 column volumes of Buffer A (8M Urea, 0.5M NaCl, 0.02M Tris, pH=7.0), collecting the effluent as equilibration solution. Then, perform gradient elution using different imidazole concentrations (10%, 20%, 30%, 50%, 100%) of Buffer B (8M Urea, 0.5M NaCl, 0.02M Tris, 0.5M Imidazole, pH=7.0), collecting the eluent for each gradient. SDS-PAGE analysis confirmed that the target protein began to elute at a 50% gradient. Figure 2 As shown, this demonstrates that the Mfp-ELP fusion protein can be expressed in a soluble manner.

[0080] Protein dialysis: The protein solution collected by affinity chromatography was placed into a 3kD dialysis bag and dialyzed for 36 hours using a 5% acetic acid dialysis buffer. Then, the protein was dialyzed with PBS buffer to remove the acetic acid and the target protein was collected.

[0081] The purified Mfp protein was prepared according to the method described in this embodiment.

[0082] Example 2

[0083] 1. In vitro cell safety test of the fusion protein Mfp-ELP

[0084] The safety of the fusion protein Mfp-ELP was investigated using human skin fibroblasts (HSF) and mouse fibroblasts (NIN / 3T3) via CCK-8 assay. HSF and NIN / 3T3 cells were removed from the liquid nitrogen container and immediately placed in a 37°C water bath, gently agitated until the cell suspension was completely thawed. The thawed cells were then transferred to a clean bench, pipette-washed thoroughly, and transferred to centrifuge tubes. Complete culture medium was added, the cells were centrifuged, and the supernatant was discarded. More culture medium was added, and the cells were thoroughly mixed. Finally, all cells were transferred to culture flasks and incubated in a sterile incubator at 37°C and 5% CO2. Subsequent experiments were performed once cell viability reached 95%.

[0085] First, the effect of the fusion protein Mfp-ELP on the viability of HSF and NIN / 3T3 cells was investigated using the CCK-8 assay. Two cell suspensions for logarithmic growth were diluted, and 5000 cells were added to each well of a 96-well plate. After culturing the cells in an incubator for 24 h, the complete medium was replaced with incomplete medium (DMEM F12 medium containing 1% penicillin and 1% streptomycin, and DMEM medium containing 1% penicillin and 1% streptomycin) containing different concentrations of the fusion protein Mfp-ELP (0 (Control), 0.1%, 0.05%, 0.025 mg / mL). After incubation in a cell culture incubator for 24 h, the medium was discarded, and 10% CCK-8 solution was added. The plates were then returned to the incubator for further incubation for 1–4 h. After incubation, the medium was removed, and the absorbance at 450 nm was immediately measured using a microplate reader. Each group was configured with three replicates. Cell viability was calculated as follows: Cell viability (%) = (Average absorbance of treatment group / Average absorbance of control group) × 100%.

[0086] The results are as follows Figure 3 As shown, the survival rates of HSF cells and NIN / 3T3 cells containing the fusion protein Mfp-ELP were not significantly different from those without the protein, indicating that the fusion protein does not have cytotoxic effects on cells.

[0087] 2. Investigation of the UV damage repair capacity of the fusion protein Mfp-ELP

[0088] Log-grown HSF cells were used at 5 × 10⁻⁶ 4Cells were seeded per well in 96-well plates and cultured for 24 h. The culture medium was discarded, and 100 μL of PBS was added. UVB radiation was then introduced, followed by discarding the PBS and adding basal medium containing either monomeric Mfp protein (0.05 mg / mL) or Mfp-ELP fusion protein (0.05 mg / mL). The plates were incubated for 24 h, then the medium was discarded, and 100 μL of 10% CCK-8 solution was added. The plates were then returned to the incubator for further incubation for 1–4 h. After incubation, the culture medium was removed, and the absorbance at 450 nm was immediately measured using a microplate reader. The treatment without UVB and protein was used as a blank control, and the treatment with UVB but without protein was used as a blank model group. Each group was replicated in triplicate.

[0089] The results are as follows Figure 4 As shown, the fusion protein Mfp-ELP has a better repair effect on UV-damaged cells than the Mfp monomer protein.

[0090] Example 3

[0091] 1. Preparation of Mfp-ELP hydrogel dressing

[0092] This system uses a PI-PAM / BACA hydrogel system: 5 mg, 10 mg, 12 mg, or 20 mg Mfp-ELP, 0.1 g AM (acrylamide), 2 mg N,N'-bis(acryloyl)cysteine ​​(BACA), and 1 mg photoinitiator (Irgacure 2959) are dissolved in 1 mL of water and gelled under UV light (365 nm) for 20 min to obtain Mfp-ELP hydrogels of different concentrations (0.5% w / v, 1.0% w / v, 1.2% w / v, 2.0% w / v). Following the same steps, 12 mg Mfp-ELP was replaced with an equal amount of MAP-Reno to obtain a MAP-Reno hydrogel with a concentration of 1.2% w / v; the hydrogel obtained without the addition of protein is recorded as the blank hydrogel.

[0093] Among them, MAP-Reno, model: MAP-Reno®M5E05C, was purchased from Shenzhen Baiyin Biotechnology Co., Ltd.

[0094] 2. The performance of Mfp-ELP hydrogels with different concentrations was investigated below.

[0095] (1) Safety test of extracellular fluid in Mfp-ELP hydrogel extraction

[0096] Mfp-ELP hydrogel extract: The prepared hydrogel was placed in the basic culture medium at a ratio of 0.1 g hydrogel / mL basic culture medium and extracted at 37℃ and 220 rpm for 48 h. The extract was then collected.

[0097] Following the preparation steps of Mfp-ELP hydrogel extract, MAP-Reno hydrogel extract and blank hydrogel extract were obtained.

[0098] The safety of the hydrogel was assessed using human skin fibroblast (HSF) cells via the CCK-8 assay. HSF cells were removed from the liquid nitrogen container and immediately placed in a 37°C water bath, gently agitated until the cell suspension was completely thawed. The thawed cells were then transferred to a clean bench, pipetted to homogenize, and transferred to centrifuge tubes. Complete culture medium was added, the tubes were centrifuged, and the supernatant was discarded. More culture medium was added, and the tubes were re-mixed. Finally, all the cells were transferred to culture flasks and incubated in a sterile incubator at 37°C and 5% CO2. Subsequent experiments were conducted once cell viability reached 95%.

[0099] First, the effect of Mfp-ELP hydrogel extract on HSF cell viability was investigated using the CCK-8 assay. Groups included: a blank control group (Control), a blank hydrogel extract group (Blank), and groups with different concentrations of Mfp-ELP hydrogel extract (0.5% Mfp-ELP, 1.0% Mfp-ELP, and 2.0% Mfp-ELP). Each group had three replicates. The two cell suspensions for logarithmic growth were diluted, and 5000 cells were added to each well of a 96-well plate. After culturing cells in the incubator for 24 hours, the complete medium (DMEMF12 medium containing 1% penicillin and 1% streptomycin) of different concentrations (0.5% w / v, 1.0% w / v, 2.0% w / v) of Mfp-ELP hydrogel extract was used to replace the complete medium, and the cells were co-cultured for another 24 hours. Then, 100 μL of CCK-8 solution was added to each well, and the plate was returned to the incubator for further incubation for 1–4 hours. After incubation, the medium was removed, and the absorbance at 450 nm was immediately measured using a microplate reader. Cell viability was calculated as: Cell viability (%) = (Average absorbance of treatment group / Average absorbance of control group) × 100%.

[0100] The results are as follows Figure 5 As shown, the survival rate of HSF cells after co-incubation with different concentrations of Mfp-ELP hydrogel extract is as high as 90% or more. The hydrogel matrix has almost no toxic effect on HSF cells and has good biocompatibility.

[0101] (2) Evaluation of ROS scavenging ability

[0102] Groups: Control group, Model group, Blank hydrogel extract group, MAP-Reno hydrogel extract group (1.2% w / v), and Mfp-ELP hydrogel extract group (1.2% w / v).

[0103] Log-grown HSF cells were used at 5 × 10⁻⁶ 4 Inoculate each well with one probe into a 96-well plate and incubate for 24 h. Incubate overnight in incomplete medium containing 0.2 mM hydrogen peroxide, then replace with medium containing hydrogel extract and co-incubate for 24 h. Gently wash with PBS, then add 100 μL of DCFH-DA probe (diluted 1000 times to 10 μM in PBS) and incubate at 37°C in the dark for 30 min. Gently wash three times with PBS, then add 100 μL of PBS and test the absorption and excitation wavelengths using a fluorescence microplate reader. The ROS scavenging rate (%) is calculated as: ROS scavenging rate (%) = [1 - (average ROS level in the treatment group / average ROS level in the model group)] × 100%.

[0104] The results are as follows Figure 6 As shown, the addition of hydrogen peroxide significantly enhanced intracellular ROS, while the addition of hydrogel extract significantly eliminated the hydrogen peroxide-induced enhancement of intracellular ROS.

[0105] (3) Hemolytic performance assessment

[0106] Groups: positive control group, blank hydrogel extract group, MAP-Reno hydrogel extract group with a concentration of 1.2% w / v (MAP-Reno), and Mfp-ELP hydrogel extract group with a concentration of 1.2% w / v (Mfp-ELP).

[0107] Whole blood was collected from the retro-orbital venous plexus of mice and collected in centrifuge tubes containing heparin sodium (10 mg / mL). The cells were centrifuged at 2000 rpm for 10 min at 4°C to collect red blood cells. The red blood cells were then repeatedly washed with 1×PBS solution by centrifugation (3000 rpm, 5 min) until no red blood was visible in the supernatant. The purified red blood cells were diluted with 1×PBS to obtain a red blood cell suspension (2%, v / v). 500 μL of the red blood cell suspension was then incubated with 500 μL of hydrogel extraction buffer, using water as a positive control and PBS as a negative control. After incubation at 37°C for 1 h, the supernatant was collected by centrifugation and added to a 96-well plate. The absorbance was measured at 540 nm.

[0108] The hemolysis rate is calculated as follows: HR(%)=[(Agel-As) / (At-As)]×100%.

[0109] Agel, At, and As represent the absorbance of each sample, positive control, and negative control at 540 nm, respectively. Experimental results are as follows: Figure 7 As shown, the hemolysis rate of the Mfp-ELP hydrogel extract is less than 5%, proving that the material does not cause hemolysis and has good biocompatibility.

[0110] 3. Animal experiments

[0111] Twenty-five male BALB / c mice (weighing 20±1g), which passed animal quarantine, were randomly divided into five groups: a blank control group (Control), a UV model group (UVB), a blank hydrogel group (Blank), a MAP-Reno hydrogel group (MAP-Reno) with a concentration of 1.2% w / v, and a Mfp-ELP hydrogel group (Mfp-ELP) with a concentration of 1.2% w / v. The mice were housed at a density of 5 mice per box under conditions of 23±1℃ and 50±5% humidity, with a 12h:12h day / night cycle of intermittent lighting. The mice had free access to food and water.

[0112] Day 1: Use a shaver to remove the fur from the animal's back. The hair removal process should be gentle to avoid damaging the skin. Day 2: Irradiate the hair-removed areas on the mouse's back with UVB (270mJ / cm²). 2 Mice were irradiated with UVB at the same time every day for 5 days, followed by drug administration according to their groups. On the seventh day after drug administration, the mice were sacrificed, photographed, and skin tissue from the dorsal modeling drug administration area was excised for HE staining and Masson staining to determine the contents of SOD, MDA, and GPX.

[0113] Results of skin damage repair ( Figure 8 The results showed that UVB irradiation causes acute inflammation and damage to the skin, and the Mfp-ELP treatment group had the best repair effect, effectively reducing the inflammatory response such as redness and damage caused by UVB, and approaching the state of healthy skin, thus verifying the application potential of this protein in the repair of skin photodamage.

[0114] HE staining revealed ( Figure 9In the control group (Control), the skin texture of mice was clear, with distinct layers, moderate epidermal thickness, and neatly and compactly arranged collagen fiber bundles in the dermis. In the model group (UVB), mice exposed to ultraviolet light showed significant uneven thickening of the epidermis, irregular proliferation of hair follicles and sebaceous glands, blurred dermal-epidermal junction, broken and loosely arranged collagen fiber bundles in the dermis, and inflammatory cell infiltration. While the MAP-Reno hydrogel group (MAP-Reno) significantly alleviated epidermal thickening, the dermal-epidermal junction remained unclear. The blank hydrogel group (Blank) showed significant epidermal thickening accompanied by severe crusting. The Mfp-ELP group of this invention exhibits clear layering of the epidermis and dermis, and can significantly alleviate uneven epidermal thickening, broken and loose collagen fiber bundles, and inflammatory cell infiltration associated with sunburn.

[0115] Masson staining revealed ( Figure 10 In the control group (Control), collagen fibers were tightly and orderly arranged in a network structure in the dermis. In the UVB model group (UVB), the collagen fiber content in the skin of mice was reduced, the distribution was disordered, and degradation, breakage, and abnormal accumulation were observed. The Mfp-ELP group of this invention showed that collagen fibers were essentially restored to a tight network structure. These experimental results indicate that the Mfp-ELP fusion protein of this invention can significantly alleviate the adverse reactions caused by ultraviolet radiation, such as collagen fiber degradation, reduced content, and uneven distribution.

[0116] The detection of the activity of MDA, a product of oxidative stress in mouse skin, revealed ( Figure 11 In the A group (UVB), the skin MDA activity of mice in the UV model group was significantly increased compared to the control group. However, compared to the UVB group, the skin MDA of mice treated with the Mfp-ELP fusion protein hydrogel of this invention was significantly reduced after UV irradiation repair. These experimental results indicate that the Mfp-ELP fusion protein of this invention can reduce endogenous oxidative stress products in the skin, thereby effectively alleviating sunburn.

[0117] The detection of the activity of antioxidant enzymes SOD and Gpx in mouse skin revealed ( Figure 11 In the UVB model group (B and C), the activities of SOD and Gpx in mouse skin were significantly reduced compared to the control group. However, compared to the UVB model group, the skin antioxidant enzymes in the mice treated with the Mfp-ELP fusion protein hydrogel of this invention significantly increased after UV irradiation repair. These experimental results indicate that the Mfp-ELP fusion protein of this invention can enhance the activity of endogenous antioxidant enzymes in the skin, thereby effectively alleviating sunburn.

[0118] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fusion protein Mfp-ELP with ultraviolet damage repair function, characterized in that, The amino acid sequence of the fusion protein Mfp-ELP is shown in SEQ ID No:

1.

2. The encoding gene of the fusion protein Mfp-ELP as described in claim 1.

3. The encoding gene according to claim 2, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID No:

2.

4. The biomaterial related to the fusion protein Mfp-ELP as described in claim 1, characterized in that: It is any one or a combination of the following biological materials: (a) An expression cassette containing the gene encoding as described in claim 2 or 3; (b) A recombinant expression vector containing the encoding gene of claim 2 or 3; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant bacteria containing the encoding gene as described in claim 2 or 3; (e) Recombinant bacteria containing the expression cassette described in (a); (f) Recombinant bacteria containing the recombinant expression vector described in (b) or (c).

5. The biomaterial according to claim 4, characterized in that: The host bacteria corresponding to the recombinant bacteria described in (d), (e), and (f) are selected from prokaryotes.

6. The biomaterial according to claim 4, characterized in that: The starting vector for the recombinant expression vectors described in (b) and (c) is a pET series vector.

7. The use of the encoding gene according to any one of claims 2 to 3 or the biomaterial according to any one of claims 4 to 6 in the preparation of the fusion protein Mfp-ELP.

8. The use of the fusion protein Mfp-ELP of claim 1, the encoding gene of any one of claims 2-3, or the biomaterial of any one of claims 4-6, mussel adhesive protein Mfp, or biomaterials related to mussel adhesive protein Mfp in the preparation of products resistant to UV damage, characterized in that: The amino acid sequence of the mussel adhesive protein Mfp is shown as 1 to 87aa in SEQ ID No: 1; The mussel adhesive protein Mfp-related biomaterial is any one or more combinations of the following biomaterials: (A) An expression cassette containing the gene encoding mussel adhesive protein Mfp; (B) A recombinant expression vector containing the gene encoding mussel adhesive protein Mfp; (C) A recombinant expression vector containing the expression cassette described in (A); (D) Recombinant bacteria containing the gene encoding mussel adhesive protein Mfp; (E) Recombinant bacteria containing the expression cassette described in (A); (F) Recombinant bacteria containing the recombinant expression vector described in (B) or (C).

9. The application according to claim 8, characterized in that: At least one of the following applications: 1) Application in the preparation of products for preventing and treating skin photoaging; 2) Application in the preparation of products for repairing UVB-induced skin damage; 3) Application in the preparation of products for adjuvant treatment of photodamaged skin diseases.

10. The application according to claim 8 or 9, characterized in that: The product includes at least one of pharmaceuticals, medical biomaterials, and cosmetics.

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