Use of silk fibroin fusion proteins in the preparation of topical medications for skin wounds

Abstract

The present invention aims to provide a use of silk fibroin fusion protein in the preparation of an external medicine for skin damage. [Solution] The silk fibroin fusion protein consists of a silk fibroin light chain linked to the neurotrophic factor NT-3 via a flexible linker chain, and self-assembles with silk fibroin to form a new material that can exert long-lasting stabilizing effects and repair skin damage. This material suppresses inflammation, promotes the deposition of type III collagen, promotes hair follicle regeneration, and enables scarless wound healing.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical materials, and in particular to silk fibroin fusion proteins with NT3 activity and the use of silk fibroin fusion proteins in skin wound repair. [Background technology]

[0002] With the development of emerging fields such as tissue engineering and biomaterials, an increasing number of biomaterials are being used to treat skin injuries. Biomaterials suitable for skin injury repair are needed to promote wound healing and reduce scar formation. Currently, many wound dressings, including hydrogels, inorganic composite microspheres, and functional nanofiber membranes, have been designed for wound healing. However, wound healing can be very time-consuming and can cause pain and further damage. Therefore, innovative bioactive materials must be designed to reduce wound pain and shorten the wound healing process. Natural biomaterials, such as chitosan, collagen, gelatin, and silk fibroin, have structures similar to the extracellular matrix, low cytotoxicity, excellent cytocompatibility, and low antigenicity. Natural biomaterials are beneficial for inducing cell proliferation, migration, and differentiation, and also possess specific recognition sites for cell surface receptors, reducing the likelihood of immune rejection. Silk fibroin is an insoluble biological protein with excellent biocompatibility in biomedical applications and has been approved by the U.S. Food and Drug Administration (FDA) as a biomaterial for human medical use. Currently, silk fibroin is widely used in tissue engineering stents due to its biocompatibility and slow biodegradability, and silk fibroin can also promote wound healing by promoting collagen remodeling.

[0003] The nerve growth factor family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophic factor-3 (NT-3), has been shown to play an important role in cardiovascular development. There are reports in the literature that intramuscular injection of NT-3 can significantly promote angiogenesis in the muscles of ischemic limbs. Angiogenesis is a critical process for promoting wound healing.

[0004] However, because NT-3 rapidly diffuses at the injury site, it is important to find an effective method to maintain an adequate concentration of NT-3. Furthermore, the instability and limited supply of the trophic factor in the body severely limit its use. Most studies on NT-3 have focused on its effects on axonal regeneration and promoting recovery after spinal cord or sciatic injuries, but its use in skin injuries has rarely been reported. Summary of the Invention [Problem to be solved by the invention]

[0005] The objective of the present invention is to provide a silk fibroin fusion protein in which neurotrophic factor NT-3 is linked to silk fibroin light chains via a flexible linker chain to form a recombinant protein, which then self-assembles with silk fibroin to form a new type of material that can exert long-lasting stabilizing effects and repair skin damage, and which can suppress inflammation, promote type III collagen deposition, promote hair follicle regeneration, and enable scarless wound healing. [Means for solving the problem]

[0006] In order to achieve the above object, the present invention provides the following technical means: 1. A silk fibroin fusion protein comprising: The silk fibroin fusion protein used herein is composed of the amino acid sequence MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT.

[0007] In the present invention, the method for preparing the protein comprises: (1) Construction of a recombinant expression vector: synthesizing a gene fragment containing silk fibroin light chain and NT-3, and ligating it into a pET-30 expression vector to complete the construction of a recombinant expression vector; and (2) Expression and purification of recombinant protein: This includes the steps of transforming the recombinant expression vector obtained in step (1) into BL21 E. coli, inducing and identifying the expression in a small test tube at 37°C, amplifying the expression, harvesting the cells, disrupting the cells by ultrasound, and purifying the recombinant protein using Ni-NTA for identification and detection.

[0008] Use of the silk fibroin fusion protein in the preparation of an external medicine.

[0009] The topical agent further contains regenerated silk fibroin.

[0010] In the present invention, the method for preparing the regenerated silk fibroin comprises: Step 1: Silkworm silk is placed in 0.5% NaHCO3 solution and boiled to remove sericin; The process includes step 2, in which the silk fibers from which sericin has been removed are washed with water, dissolved in a mixed solution of CaCl2, H2O and C2H5OH (molar ratio 1:8:2), then dialyzed against distilled water (molecular weight cut-off of dialysis bag: 12,000-14,000 Da), and freeze-dried to obtain regenerated silk fibroin.

[0011] An external preparation comprising the silk fibroin fusion protein and regenerated silk fibroin.

[0012] In addition, in the topical preparation, the silk fibroin fusion protein is immobilized on the regenerated silk fibroin by self-assembly with the regenerated silk fibroin.

[0013] In addition, in the topical preparation, the blending ratio of regenerated silk fibroin to silk fibroin fusion protein is 1 mL of 0.2 g / mL regenerated silk fibroin solution to 2 μL of 100 μg / mL silk fibroin fusion protein solution.

[0014] Use of the above topical preparation in the preparation of a scarless skin wound healing promoter.

[0015] Use of the above topical preparation in the preparation of an agent for suppressing inflammation of skin wounds.

[0016] Use of the above topical formulation in the preparation of a hair follicle regeneration promoter.

[0017] The silk fibroin fusion protein and regenerated silk fibroin of the present invention can be applied as a solution or mixed with a dressing commonly used for skin injuries to treat skin injuries. The mixed solution can also be dried in a refrigerator at 4°C to form a film, resulting in a silk fibroin film with NT-3 activity, which can then be processed into gauze or bandages for wound treatment. [Effects of the Invention]

[0018] The main materials used in this invention are silk fibroin and silk fibroin light chains fused with NT-3, and they have good biocompatibility because no toxic or side-effect substances such as crosslinkers or surfactants are added during the preparation process.

[0019] The skin damage repair material provided by the present invention introduces a silk fibroin light chain fused with NT-3, maintains the disulfide bond of the light chain, and adds a flexible linker chain between NT3 and the light chain. In the silk fibroin solution, the silk fibroin self-assembles, and NT-3 is fixed to the silk fibroin together with the silk fibroin light chain that maintains the disulfide bond. The flexible chain of FIBL-Linker-NT3 serves to link NT3 to other NT3 in different directions.

[0020] During the repair process of skin injury, the wound recovery state was observed, and after treatment with silk fibroin and silk fibroin light chain fused with NT-3, it was found that it significantly promoted wound recovery, inhibited wound inflammation, promoted type III collagen deposition, increased blood vessel area, epithelial length, and the number of regenerating hair follicles, and promoted scarless skin wound healing. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows a schematic diagram of the recombinant protein FIBL-Linker-NT3 and the results of Western blot analysis. [Figure 2] The left figure shows the results of silver staining of silk fibroin (SF) and silk fibroin heavy chain (SFH), and the right figure shows the results of Western blot of SFH-FIBL-Linker-NT3. [Figure 3] FIG. 1 shows the experimental results of Example 2 showing that recombinant proteins promote the recovery of skin damage in mice. [Figure 4] FIG. 1 shows experimental results of inflammation of mouse skin wounds in Example 3. [Figure 5] FIG. 1 shows the experimental results of type III collagen deposition in mouse skin wounds in Example 4. [Figure 6]FIG. 1 shows the experimental results of Example 5 regarding vascularization of mouse skin wounds, the length of regenerated epithelium, and the number of regenerated hair follicles. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following examples are provided to further illustrate preferred embodiments of the present invention. It should be understood that the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and scope of the present invention.

[0023] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0024] Materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0025] (Example) Example 1 1. Expression and verification of FIBL-Linker-NT3 recombinant protein (1) A recombinant expression vector was constructed, the codons for the silk fibroin light chain and NT3 active peptide were optimized, and a flexible linker chain was added to synthesize the gene. The synthesized gene was then constructed into the vector pET-30a by enzymatic digestion and ligation, and the successful construction of the recombinant expression vector pET-FIBL-Linker-NT3 was verified by sequencing.

[0026] (2) The recombinant vector was transformed into BL21 E. coli to construct an expression strain. A single clone was collected in 1 mL of LB liquid medium and cultured overnight at 37°C and 150 rpm. Expression was identified and the expression strain was preserved. The next day, the culture was increased to 50 mL and shaken in a shaker at 37°C until the OD600 of the culture reached approximately 1.0. IPTG was added to a final concentration of 1 mM and expression was induced at 37°C for 16 hours.

[0027] (3) The bacterial precipitate was collected by centrifugation. After ultrasonic disruption, the His-tagged target protein was separated and purified using Ni-NTA, and the expression of the target protein FIBL-Linker-NT3 was identified by SDS-PAGE and Western blot.

[0028] The results are shown in Figure 1, indicating the successful construction and expression of the fusion protein FIBL-Linker-NT3.

[0029] II. Silk fibroin can self-assemble with FIBL-Linker-NT3 (1) 0.1 g of silk fibroin was dissolved in 2 mL of PBS (0.1 M) at pH 7.4, 100 μL of DTT (1 M) was added, sealed, and reacted at 4 °C for 6 hours. The solution was placed in a cellulose tube (molecular cutoff value 100 kDa) and immersed in distilled water for 3 days. The deionized water was changed once every 8 - 10 hours. The silk fibroin heavy chain (SFH) in the cellulose tube was recovered, and together with the original silk fibroin, it was verified by silver staining that the separation of the silk fibroin heavy chain (SFH) was successful.

[0030] (2) 100 μL of silk fibroin heavy chain SFH (7.5 μg / μL) was added to 9.9 mL of PBS (0.1 M) at pH 7.4, and then 180 μL of recombinant protein FIBL-Linker-NT3 (100 μg / mL) was added for self-assembly. It was placed in a 100 kDa cellulose tube and dialyzed for 3 days. The SFH-FIBL-Linker-NT3 in the cellulose tube was reserved for further Western blot analysis. When detected with the NT3 antibody, it was found that the molecular band of the self-assembled SFH-FIBL-Linker-NT3 was above 100 kDa, proving the success of self-assembly. The results are shown in Figure 2.

[0031] (Example 2) <The <SF+FIBL-Linker-NT3> recombinant protein solution promotes the recovery of mouse skin injury> (1) Blank group: Eight-week-old male adult mice were purchased from the Experimental Animal Center of Nantong University and anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution. A 10 mm diameter circular wound was created on the back of each mouse using a sterile punch. 100 μL of PBS (0.1 M, pH 7.4) was instilled daily.

[0032] (2) 50 g of mulberry silk was placed in 2 L of boiling 0.5% NaHCO3 solution and boiled for 30 minutes. The water was then discarded, the sericin was removed, and the silk was washed three times with triple-distilled water. This was repeated three times, and the silk was then placed in a clean bench and air-dried to obtain silk fibroin fibers with the outer sericin removed.

[0033] (3) Experimental Group 1: 20 g of the obtained silk fibroin fiber was dissolved in a ternary copolymer solvent system of CaCl2 / H2O / C2H5OH (molar ratio 1:8:2). The resulting silk fibroin solution was then dialyzed against triple-distilled water. The molecular weight cutoff of the dialysis bag was in the range of 12,000–14,000 Da. After dialysis at 4°C for 3 days, the silk fibroin was freeze-dried in a freeze dryer to obtain regenerated silk fibroin. 0.2 g of regenerated silk fibroin was weighed and dissolved in 1 mL of 0.1 M PBS, pH 7.4, to obtain a 20% silk fibroin solution. A mouse skin injury model was obtained using the blank group method, and 100 μL of silk fibroin solution (SF) was instilled daily.

[0034] (4) Experimental group 2: 20 μL of recombinant protein FIBL-Linker-NT3 (100 μg / mL) was added to 1 mL of PBS (0.1 M), pH 7.4. A mouse skin injury model was obtained by following the method of the blank group. 100 μL of FIBL-Linker-NT3 recombinant protein solution was instilled daily.

[0035] (5) Experimental group 3: 0.2 g of regenerated silk fibroin and 2 μL of recombinant protein FIBL-Linker-NT3 (100 μg / mL) were weighed and added to 1 mL of PBS (0.1 M) at pH 7.4. A mouse skin injury model was obtained by following the method of the blank group, and 100 μL of SF+FIBL-Linker-NT3 recombinant protein solution was dripped into the skin every day.

[0036] (6) On the 0th, 5th, and 10th days after skin injury, the skin injury on the back of the mice was photographed and observed. As shown in Figure 3, SF+FIBL-Linker-NT3 was helpful for the recovery of the skin injury on the back of the mice.

[0037] (Example 3) <SF+FIBL-Linker-NT3 recombinant protein thin film promotes the recovery of mouse skin injury> (1) 50 g of mulberry silk raw silk was put into 2 L of 0.5% NaHCO3 boiling solution, boiled for 30 minutes, then the water was discarded to remove sericin, washed 3 times with triple distilled water, repeated a total of 3 times, put into a clean bench and dried by blowing to obtain silk fibroin fibers with the outer sericin removed.

[0038] (2) Control group: 20 g of the obtained silk fibroin fibers were dissolved in a ternary copolymer solvent system of CaCl2 / H2O / C2H5OH (molar ratio 1:8:2). Then, the obtained silk fibroin solution was dialyzed with triple distilled water, and the molecular weight cut-off of the dialysis bag was in the range of 12000 - 14000 Da. After dialysis at 4°C for 3 days, it was freeze-dried on a freeze dryer to obtain regenerated silk fibroin. 0.2 g of regenerated silk fibroin was weighed and dissolved in 1 mL of PBS (0.1 M) with pH 7.4 to obtain a 20% silk fibroin solution. A sterilized small round slide glass with a diameter of 14 mm was placed in a 24-well plate, 100 μL of the silk fibroin solution was dropped onto the small round slide glass, and it was placed in a refrigerator at 4°C and dried overnight to form a film. 8-week-old male adult mice were purchased from the Experimental Animal Center of Nantong University and anesthetized by intraperitoneal injection of 3% pentobarbital sodium solution. Using a sterilized punch, a circular wound with a diameter of 10 mm was created on the back of each mouse. The silk fibroin thin film was covered on the wound and wrapped with gauze.

[0039] (3) Experimental group: 0.2 g of regenerated silk fibroin and 2 μL of recombinant protein FIBL-Linker-NT3 (100 μg / mL) were weighed and added to 1 mL of PBS (0.1 M) at pH 7.4. After establishing a skin injury model in mice, an SF + FIBL-Linker-NT3 thin film was covered on the wound and wrapped with gauze.

[0040] (4) On the 0th, 5th, and 10th days after skin injury, the skin injury on the back of the mice was photographed and observed. The results showed that the SF + FIBL-Linker-NT3 thin film was helpful for the recovery of the skin injury on the back of the mice compared with the single silk fibroin thin film.

[0041] (Example 4) <SF + FIBL-Linker-NT3 reduces wound inflammation in mice> (1) Referring to Example 2, a skin injury model on the back of the mice was established.

[0042] (2) Five days after skin injury, the skin tissue on the back of the mice was collected, fixed with 4% paraformaldehyde, dehydrated with 30% sucrose after 24 hours, and then frozen sections with a thickness of 12 μm were prepared.

[0043] (3) After section preparation, immunofluorescence staining was performed, inflammatory cells were labeled with iNOS, and photographs were taken with a fluorescence microscope to observe the inflammatory state of the injury site.

[0044] (4) The results are shown in Figure 4. The inflammation in the SF group and the SF + FIBL-Linker-NT3 group was significantly reduced compared with the PBS and PBS + FIBL-Linker-NT3 groups, indicating that silk fibroin can inhibit the inflammation at the wound site.

[0045] (Example 5) <The SF + FIBL-Linker-NT3 group significantly promotes the deposition of type III collagen> (1) Referring to Example 2, a skin injury model on the back of the mice was established.

[0046] (2) Five days after the skin injury, the skin tissue on the back of the mouse was collected, fixed with 4% paraformaldehyde, dehydrated with 30% sucrose 24 hours later, and then frozen sections with a thickness of 12 μm were prepared.

[0047] (3) After section preparation, immunofluorescence staining was performed. Type III collagen was labeled with Col3, and photographs were taken with a fluorescence microscope to observe the deposition state of type III collagen.

[0048] (4) The results are shown in Figure 5. The deposition of type III collagen in the SF+FIBL-Linker-NT3 group was significantly more than that in the other three groups, indicating that when silk fibroin and FIBL-Linker-NT3 are present, the deposition of type III collagen can be significantly promoted, and scar formation can be reduced.

[0049] (Example 6) <The SF+FIBL-Linker-NT3 group significantly promotes angiogenesis, the length of the regenerated epithelium, and the number of regenerated hair follicles> (1) Referring to Example 2, a skin injury model on the back of the mouse was prepared.

[0050] (2) Five days after the skin injury, the skin tissue on the back of the mouse was collected, fixed with 4% paraformaldehyde, dehydrated with 30% sucrose 24 hours later, and then frozen sections with a thickness of 12 μm were prepared.

[0051] (3) After section preparation, hematoxylin-eosin (HE) staining was performed. The hematoxylin staining solution is alkaline and mainly stains the chromatin in the nucleus and ribosomes in the cytoplasm purple-blue. Eosin is an acidic dye and mainly stains the components in the cytoplasm and extracellular matrix red. The results are shown in Figure 6. The SF+FIBL-Linker-NT3 group can effectively promote angiogenesis and increase the length of the regenerated epithelium and the number of regenerated hair follicles.

[0052] (Supplementary Note) (Supplementary Note 1) 1. Use of a silk fibroin fusion protein in the preparation of a topical medicament for skin damage, the silk fibroin fusion protein comprising: Use characterized in that the amino acid sequence is represented by MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT.

[0053] (Appendix 2) The use described in Appendix 1, characterized in that the topical skin damage medication further contains regenerated silk fibroin.

[0054] (Appendix 3) The method for preparing regenerated silk fibroin comprises: Step 1: Silkworm silk is placed in 0.5% NaHCO3 solution and boiled to remove sericin; After washing the silk fibers from which sericin has been removed with water, they are dissolved in a mixed solution of CaCl2, H2O, and C2H5OH (molar ratio 1:8:2), then dialyzed against distilled water (molecular weight cutoff of dialysis bag: 12,000 to 14,000 Da), and freeze-dried to obtain regenerated silk fibroin. 2. The use according to claim 1, comprising:

[0055] (Appendix 4) 1. A topical formulation comprising a silk fibroin fusion protein and regenerated silk fibroin, wherein the silk fibroin fusion protein comprises: An external preparation characterized by consisting of an amino acid sequence represented by the amino acid sequence MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT.

[0056] (Appendix 5) The topical preparation according to claim 4, wherein the silk fibroin fusion protein is immobilized on the regenerated silk fibroin by self-assembly with the regenerated silk fibroin.

[0057] (Appendix 6) Use of the topical formulation described in Appendix 4 in the preparation of a scarless skin wound healing promoter.

[0058] (Appendix 7) Use of the topical formulation described in Appendix 4 in the preparation of a skin wound inflammation inhibitor.

[0059] (Appendix 8) Use of the topical formulation described in Appendix 4 in the preparation of a hair follicle regeneration promoter.

Claims

1. 1. Use of a silk fibroin fusion protein in the preparation of a topical medicament for skin damage, the silk fibroin fusion protein comprising: MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAAANVINSYT DGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAH Use characterized in that it consists of an amino acid sequence represented by LLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCCVCALSRKIGRT.

2. The use according to claim 1, characterized in that the topical skin injury medication further comprises regenerated silk fibroin.

3. The method for preparing regenerated silk fibroin comprises: Silkworm silk was diluted with 0.5% NaHCO ３ Step 1: Putting the mixture into a solution and boiling it to remove sericin; The silk fibers from which sericin had been removed were washed with water, and then treated with CaCl ２ , H ２ O and C ２ H ５ Step 2: Dissolve the silk fibroin in a mixed solution of hydroxypropyltrimonials (OH) (molar ratio 1:8:2), then dialyze it against distilled water (molecular weight cut-off of dialysis bag: 12,000-14,000 Da), and freeze-dry it to obtain regenerated silk fibroin.

2. The use according to claim 1, characterized in that it comprises:

4. 1. A topical formulation comprising a silk fibroin fusion protein and regenerated silk fibroin, wherein the silk fibroin fusion protein comprises: MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAAANVINSYT DGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHL 1. An external preparation comprising an amino acid sequence represented by the formula: LPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCCVCALSRKIGRT.

5. The topical preparation according to claim 4, wherein the silk fibroin fusion protein is immobilized on the regenerated silk fibroin by self-assembly with the regenerated silk fibroin.

6. Use of the topical preparation according to claim 4 in the preparation of a scarless skin wound healing promoter.

7. Use of the topical preparation according to claim 4 in the preparation of an agent for suppressing inflammation of skin wounds.

8. Use of the topical formulation according to claim 4 in the preparation of a hair follicle regeneration promoter.

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