CN117100840A - Application of silk fibroin fusion protein in the preparation of external products for skin damage - Google Patents

Abstract

The invention discloses the application of silk fibroin fusion protein in the preparation of external products for skin damage. The silk fibroin fusion protein is composed of neurotrophic factor NT-3 and silk fibroin light chain combined through a flexible linker chain. The silk fibroin fusion protein can self-assemble with silk fibroin to form a stable protein that can function for a long time. It is a new material that can repair skin damage. This material can inhibit inflammation, promote the deposition of type III collagen, promote the regeneration of hair follicles, and enable scar-free wound healing.

Description

Application of silk fibroin fusion protein in the preparation of external products for skin damage

Technical field

The invention belongs to the field of biomedical materials, and specifically relates to a silk fibroin fusion protein with NT3 activity and its application in skin damage repair.

Background technique

With the development of emerging disciplines such as tissue engineering and biomaterials, more and more biomaterials are used in skin injuries. Suitable biomaterials for repairing skin damage should 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 is a very time-consuming process that can cause pain and further damage. In order to reduce wound pain and speed up the wound healing process, it is necessary to design innovative bioactive materials. Natural biological materials, such as chitosan, collagen, gelatin, silk fibroin, etc., all have structures similar to extracellular matrix. They not only have low cytotoxicity, good cell compatibility, but also have low antigenicity. Natural biomaterials are beneficial to inducing cell proliferation, migration and differentiation. In addition, because they have specific recognition sites for cell surface receptors, they are not prone to immune rejection. Silk fibroin is an insoluble biological protein with good biocompatibility in biomedical applications. It has been approved by the U.S. Food and Drug Administration (FDA) as a human medical biomaterial. Currently, silk fibroin is widely used in tissue engineering scaffolds due to its biocompatibility and slow biodegradability. In addition, silk fibroin can also accelerate wound healing by promoting the reorganization of collagen.

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 a key role in cardiovascular development. There are reports in the literature that intramuscular injection of NT-3 can significantly promote angiogenesis in ischemic limb muscles. Angiogenesis is a critical step in promoting wound healing.

However, NT-3 diffuses rapidly at the injury site, and it is crucial to find an effective method to maintain NT-3 at an appropriate concentration. At the same time, the instability of nutritional factors in the body and their limited sources have greatly restricted their use. And most of the research on NT-3 focuses on its effect on neuronal axon regeneration and promoting the recovery of spinal cord injury or ischial injury. There are few reports on its effect on skin damage.

Contents of the invention

The purpose of the present invention is to provide a silk fibroin fusion protein that combines neurotrophic factor NT-3 with silk fibroin light chains through a flexible linker chain to form a recombinant protein. This recombinant protein can self-assemble with silk fibroin to form a A new material that can stabilize skin damage for a long time. This material can inhibit inflammation, promote the deposition of type III collagen, promote the regeneration of hair follicles, and enable scar-free wound healing.

In order to achieve the above objects, the present invention adopts the following technical solutions:

A silk fibroin fusion protein whose amino acid sequence is:

MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDL LRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT (seq_1).

In the present invention, the preparation method of the above-mentioned protein is:

(1) Construction of recombinant expression vector: Synthesize the gene fragment containing silk fibroin light chain and NT-3, connect it to the pET-30 expression vector, and complete the construction of the recombinant expression vector.

(2) Expression and purification of recombinant protein: Transfer the recombinant expression vector obtained in step (1) into BL21 E. coli, induce expression in small test tubes at 37°C and identify, then amplify expression and collect cells, sonicate, and use Ni-NTA to purify and recombinant Protein identification and detection.

Application of the above-mentioned silk fibroin fusion protein in the preparation of external products.

Further, the external product also includes regenerated silk fibroin.

In the present invention, the preparation method of the regenerated silk fibroin is:

Step 1: Boil mulberry silk in 0.5% NaHCO ₃ solution to degumm;

Step 2: Wash the degummed silk fiber with water, dissolve it in a mixture of CaCl ₂ , H ₂ O and C ₂ H ₅ OH (molar ratio 1:8:2), and then dialyze with distilled water. The molecular weight cutoff of the dialysis bag is about 12000-14000 Da, regenerated silk fibroin is obtained after freeze-drying.

Further, in the external preparation, the dosage ratio of regenerated silk fibroin and silk fibroin fusion protein is: add 2 μL of 100 μg/mL silk fibroin fusion protein solution to every 1 mL of 0.2 g/mL regenerated silk fibroin solution.

An external preparation, including the above-mentioned silk fibroin fusion protein and regenerated silk fibroin.

The silk fibroin fusion protein and regenerated silk fibroin of the present invention can be directly applied after being made into a solution or mixed with dressings commonly used for skin injuries to treat skin injuries. The mixed solution can also be dried in a four-degree refrigerator to form a film. The silk fibroin film with NT-3 activity is obtained, and then made into gauze or band-aid for wound treatment.

beneficial effects

The main materials used in the present invention are silk fibroin and silk fibroin light chain fused with NT-3. During the preparation process, no toxic or side-effect substances such as cross-linking agents and surfactants are added, so it has good of biocompatibility.

The skin damage repair material provided by the present invention introduces a silk fibroin light chain fused with NT-3, retains the disulfide bond of the light chain, and adds a flexible linker chain between NT3 and the light chain. Silk fibroin is self-assembling in the silk fibroin solution, and NT-3 will be fixed in the silk fibroin along with the silk fibroin light chain that retains the disulfide bond, and the flexible chain of FIBL-Linker-NT3 is conducive to the interaction between NT3 and Other NT3s bind in different directions.

In the process of repairing skin damage, through the observation of wound recovery, treatment with silk fibroin and silk fibroin light chain fused with NT-3 has been shown to significantly promote wound recovery, inhibit inflammation at the wound, and promote the deposition of type III collagen. Increase blood vessel area, epithelial length and number of new hair follicles, and promote skin damage to heal without scars.

Description of drawings

Figure 1 shows the schematic diagram and Western blot verification results of the recombinant protein FIBL-Linker-NT3.

The left picture in Figure 2 shows the silver staining results of silk fibroin (SF) and silk fibroin heavy chain (SFH), and the right picture shows the Western blot results of SFH-FIBL-Linker-NT3.

Figure 3 is the experimental results of the recombinant protein in Example 2 promoting the recovery of mouse skin damage.

Figure 4 is the experimental results of mouse skin wound inflammation in Example 3.

Figure 5 is the experimental results of type III collagen deposition in mouse skin wounds in Example 4.

Figure 6 is the experimental results of angiogenesis, new epithelial length and new hair follicle number in mouse skin wounds in Example 5.

Implementation

The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are given 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 purpose and spirit of the present invention.

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

Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1

1. Expression and verification of FIBL-Linker-NT3 recombinant protein

(1) Construct a recombinant expression vector, optimize the codons of the silk fibroin light chain and NT3 active peptide, and add a flexible linker chain in the middle, then synthesize the gene, and construct the synthesized gene into the vector pET- 30a, sequencing verified the successful construction of the recombinant expression vector pET-FIBL-Linker-NT3.

(2) Transform the recombinant vector into BL21 E. coli to construct an expression strain, pick a single clone into 1 mL LB liquid medium, culture it overnight at 37°C and 150 rpm, identify the expression, and save the expression strain. The next day, expand the culture to 50 mL, shake it on a 37°C shaker until the OD600 of the bacterial solution is about 1.0, add IPTG with a final concentration of 1 mM, and induce expression at 37°C for 16 h.

(3) Collect the bacterial sediment by centrifugation, use Ni-NTA to separate and purify the target protein with His tag after ultrasonic disruption, and identify the expression of the target protein FIBL-Linker-NT3 by SDS-PAGE and Western blot.

The results are shown in Figure 1, indicating that the fusion protein FIBL-Linker-NT3 was successfully constructed and expressed.

2. Silk fibroin can self-assemble with FIBL-Linker-NT3

(1) Dissolve 0.1 g of silk fibroin in 2 mL of PBS (0.1 M) with pH 7.4, add 100 μL of DTT (1 M), seal the bottle, and react at 4°C for 6 hours. The solution was placed in a cellulose tube (molecular cutoff value of 100 kDa) and soaked in distilled water for 3 days. Deionized water is replaced approximately every 8-10 hours. The silk fibroin heavy chain (SFH) in the cellulose tube was collected, and together with the protofilament protein, silver staining verified the successful isolation of the silk fibroin heavy chain (SFH).

(2) Add 100 μL silk fibroin heavy chain SFH (7.5 μg/μL) to 9.9 mL pH7.4 PBS (0.1 M), and then add 180 μL recombinant protein FIBL-Linker-NT3 (100 μg/mL) for self-assembly. Place in 100 kDa cellulose tube for dialysis for 3 days. The SFH-FIBL-Linker-NT3 in the cellulose tube was retained for further Western blot analysis and detected with NT3 antibody. It was found that the molecular band of self-assembled SFH-FIBL-Linker-NT3 was above 100 kDa, proving that the self-assembly was successful. The results are shown in Figure 2.

Example 2

SF+FIBL-Linker-NT3 recombinant protein solution promotes skin injury recovery in mice

(1) Blank group: 8-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 sterile punch was used to create a 10 mm diameter hole on the back of each mouse. Round wound. Add 100 μL of PBS (0.1 M) at pH 7.4 dropwise every day.

(2) Take 50 g of mulberry silkworm silk and put it into 2 L of boiled 0.5% NaHCO ₃ solution. After boiling for 30 minutes, pour out the water, remove the sericin, and wash it 3 times with three-distilled water. Repeat 3 times in total. Put it into a clean bench and blow dry to obtain the silk fibroin fiber with the outer sericin removed.

(3) Experimental group one, dissolve 20 g of the silk fibroin fiber obtained in the terpolymer solvent system of CaCl ₂ /H ₂ O/C ₂ H ₅ OH (molar ratio 1:8:2), and then obtain The silk fibroin solution is dialyzed with tri-distilled water, and the molecular weight cutoff of the dialysis bag is approximately 12,000 - 14,000 Da. After dialysis at 4°C for 3 days, it was freeze-dried on a freeze-drying machine to obtain regenerated silk fibroin. Weigh 0.2 g of regenerated silk fibroin and dissolve it in 1 mL of PBS (0.1 M) with pH 7.4 to obtain a 20% silk fibroin solution. The mouse skin injury model was obtained according to the method of the blank group, and 100 μL silk fibroin solution (SF) was added dropwise every day.

(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) with pH 7.4, and the mouse skin injury model was obtained by referring to the method of the blank group. Injections were added every day. Add 100 μL FIBL-Linker-NT3 recombinant protein solution.

(5) Experimental group three: Weigh 0.2 g of regenerated silk fibroin and 2 μL of recombinant protein FIBL-Linker-NT3 (100 μg/mL) and add it to 1 mL of PBS (0.1 M) with pH 7.4. Refer to the blank group to obtain mice. For the skin injury model, 100 μLSF+FIBL-Linker-NT3 recombinant protein solution was added dropwise every day.

(6) Take photos and observe the skin damage on the back of mice on days 0, 5, and 10 after skin injury. As shown in Figure 3, SF+FIBL-Linker-NT3 is more conducive to the recovery of mouse back skin injury.

Example 3

SF+FIBL-Linker-NT3 recombinant protein film promotes skin injury recovery in mice

(1) Take 50 g of mulberry silkworm raw silk and put it into 2 L of boiled 0.5% NaHCO ₃ solution. After boiling for 30 minutes, pour out the water, remove the sericin, and wash it 3 times with three-distilled water. Repeat 3 times in total. Put it into a clean bench and blow dry to obtain the silk fibroin fiber with the outer sericin removed.

(2) In the control group, 20 g of the obtained silk fibroin fiber was dissolved in the terpolymer solvent system of CaCl ₂ /H ₂ O/C ₂ H ₅ OH (molar ratio 1:8:2), and then the obtained The silk fibroin solution is dialyzed with triple distilled water, and the molecular weight cutoff of the dialysis bag is approximately 12,000 - 14,000 Da. After dialysis at 4°C for 3 days, it was freeze-dried on a freeze-drying machine to obtain regenerated silk fibroin. Weigh 0.2 g of regenerated silk fibroin and dissolve it in 1 mL of PBS (0.1 M) with pH 7.4 to obtain a 20% silk fibroin solution. Place a sterile small round glass slide with a diameter of 14 mm in a 24-well plate, drop 100 μL of silk fibroin solution on the small round glass slide, and place it in the refrigerator for 4 days to dry overnight to form a film. Eight-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. A 10 mm diameter circular wound with a diameter of 10 mm was made on the back of each mouse using a sterile punch. Apply silk fibroin film to the wound and wrap it with gauze.

(3) Experimental group, weigh 0.2 g of regenerated silk fibroin and 2 μL of recombinant protein FIBL-Linker-NT3 (100 μg/mL) and add it to 1 mL of PBS (0.1 M) with pH 7.4 to prepare a mouse skin injury model. Apply SF+FIBL-Linker-NT3 film to the wound and wrap it with gauze.

(4) The skin injuries on the back of mice were photographed and observed on days 0, 5, and 10 after skin injury. The results showed that the SF+FIBL-Linker-NT3 film was more beneficial to the back skin injury of mice than the monofilament film. of recovery.

Example 4

SF+FIBL-Linker-NT3 reduces wound inflammation in mice

(1) Refer to Example 2 to prepare a mouse back skin injury model.

(2) Take the mouse back skin tissue 5 days after skin injury, fix it with 4% paraformaldehyde, dehydrate it with 30% sucrose 24 hours later, and then perform frozen sectioning with the section thickness set to 12 μm.

(3) Immunofluorescence staining was performed after sectioning, iNOS was used to mark inflammatory cells, and the inflammation in the injured area was observed by taking pictures under a fluorescence microscope.

(4) The results are shown in Figure 4. The inflammation in the SF group and the SF+FIBL-Linker-NT3 group was significantly less than that in the PBS and PBS+FIBL-Linker-NT3 groups, indicating that silk fibroin can inhibit inflammation in the wound.

Example 5

The SF+FIBL-Linker-NT3 group significantly promoted type III collagen deposition.

(1) Refer to Example 2 to prepare a mouse back skin injury model.

(2) Five days after skin injury, the mouse back skin tissue was taken, fixed with 4% paraformaldehyde, dehydrated with 30% sucrose 24 hours later, and then frozen sectioned, with the section thickness set to 12 μm.

(3) After sectioning, perform immunofluorescence staining, use Col3 to label type III collagen, and take photos under a fluorescence microscope to observe the deposition of type III collagen in the injured area.

(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 of the other three groups, proving that the presence of silk fibroin and FIBL-Linker-NT3 can significantly promote the deposition of type III collagen. Reduce scarring.

Example 6

The SF+FIBL-Linker-NT3 group significantly promoted angiogenesis, new epithelial length and the number of new hair follicles.

(1) Refer to Example 2 to prepare a mouse back skin injury model.

(2) On the 5th day after skin injury, the mouse back skin tissue was taken, fixed with 4% paraformaldehyde, dehydrated with 30% sucrose 24 hours later, and then frozen sectioned, with the section thickness set to 12 μm.

(3) After sectioning, perform hematoxylin-eosin (HE) staining. The hematoxylin stain is alkaline and mainly colors the chromatin in the nucleus and the ribosomes in the cytoplasm purple-blue; eosin is an acidic dye. , mainly coloring 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, increase the length of regenerated epithelium and the number of new hair follicles.

Claims (4)

1. The application of the silk fibroin fusion protein in preparing skin injury external products is characterized in that the silk fibroin fusion protein has the amino acid sequence as follows:

MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT。

2. the use according to claim 1, wherein the skin lesion topical product further comprises regenerated silk fibroin.

3. The use according to claim 1, wherein the regenerated silk fibroin is prepared by the following method:

step 1, taking mulberry silk with 0.5% NaHCO ₃ Boiling and degumming in the solution;

step 2, washing the degummed silk fiber with water, and dissolving in CaCl ₂ 、H ₂ O and C ₂ H ₅ In the mixed solution of OH, caCl in the mixed solution ₂ 、H ₂ O and C ₂ H ₅ OH molar ratio is 1:8:2, then distilled water is used for dialysis, the cut-off molecular weight of a dialysis bag is 12000-14000 Da, and the regenerated silk fibroin is obtained after freeze drying.

4. An external preparation, comprising a silk fibroin fusion protein and regenerated silk fibroin, wherein the amino acid sequence of the silk fibroin fusion protein is as follows:

MHHHHHHAPSVTINQYSDNEIPRDIDDGKASSVISRAWDYVDDTDKSIAILNVQEILKDMASQGDYASQASAVAQTAGIIAHLSAGIPGDACAAANVINSYTDGVRSGNFAGFRQSLGPFFGHVGQNLNLINQLVINPGQLRYSVGPALGCAGGGRIYDFEAAWDAILASSDSSFLNEEYCIVKRLYNSRNSQSNNIAAYITAHLLPPVAQVFHQSAGSITDLLRGVGNGNDATGLVANAQRYIAQAASQVHVGGGGSGGGGSYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETRCKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCALSRKIGRT。