A recombinant keratin and its application

Abstract

The present application provides a recombinant keratin. When the keratin of the present application is used for wound repair, the repair function is greatly improved, especially for the repair of chronic wounds such as diabetic foot ulcers, bedsores, and venous ulcers. It accelerates the formation of new granulation and epithelial tissue during the granulation proliferation stage and epithelial formation stage, can quickly promote wound healing, promote wound repair, and has hemostatic and anti-inflammatory functions.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a recombinant keratin and an application thereof. Background Art

[0002] Peripheral angiogenesis disorders, immune deficiency, metabolic disorders, local infection, etc. in diabetic wounds can cause the epidermis, dermis, subcutaneous fat, muscle, fascia and other tissues to be in a state of damage for a long time. Therefore, inhibiting infection, improving blood circulation, and restoring immune and metabolic functions are the key to promoting the regeneration and repair of damaged tissues and accelerating wound healing. Clinicians often perform surgery or negative pressure wound therapy to promote wound healing. Traditional surgical interventions, such as debridement, skin grafts and flaps, are commonly used to treat large wounds, but they are limited by strict indications, susceptibility to infection and professional medical care requirements. Negative pressure therapy can only effectively treat very small chronic wounds, although it can accelerate the healing of chronic wounds through exudate drainage, improved microcirculation and granulation growth. In contrast, wound dressings have much fewer limitations and can be used to treat any form of wound and any stage of wound healing. Tissue biocompatibility, biodegradability, mechanical durability and natural abundance are the excellent properties of keratin biomaterials, making them dressings for the treatment of acute and chronic wounds. Human hair keratin has abundant cell-binding motifs, such as arginine-glycine-aspartic acid (RGD), leucine-aspartic acid-valine (LDV), and glutamic acid-aspartic acid-serine (EDS), which are beneficial for cell attachment and proliferation. Studies have shown that keratin plays an important role in wound hemostasis, inflammation, proliferation, and remodeling. In addition, it has been developed as an effective biomaterial to promote bone regeneration, hemostasis, and peripheral nerve repair. Since human hair keratin itself is a member of the intermediate filament family of epidermal appendages, the skin epidermis contains a large amount of epithelial keratin related to wound repair, which is highly homologous to human hair keratin. Therefore, low immunogenicity and strong biocompatibility are favorable factors for keratin to promote skin wound repair. Therefore, keratin-based wound dressings are often designed in various forms to meet the treatment needs of different wounds.

[0003] Some researchers have conducted experiments on cell adhesion on keratin hydrogels, and the results show that keratin has ligand-like interactions with at least β1 and β3 integrin subunits, which can enhance the adhesion and proliferation of cells on keratin-based materials. We have reported the wound healing properties of 17 human hair keratins in terms of wound closure rate, epidermal regeneration and angiogenesis. Among them, recombinant keratin (RK) 33B, RK39 and RK84 showed the highest efficiency in accelerating wound closure, while RK34, RK37 and RK81 showed the strongest angiogenic properties. In terms of anti-inflammatory, keratin exhibits unique anti-inflammatory properties. qRT-PCR analysis showed that 17 recombinant keratins had different activities on microglia / macrophage phenotype regulation, and confirmed that RK33A had a significant effect on the transition of microglia from M1 phenotype to M2 phenotype. The implantation of recombinant keratin 33A nanofibers is beneficial to relieve inflammation, reduce glial scar formation, improve nerve regeneration, and thus promote the recovery of motor function in the rat T9 spinal cord hemisection model. In addition, keratin can also aggregate red blood cells and rapidly coagulate blood. Aggregating red blood cells can make keratin-based dressings better bonded to the wound; and keratin-based dressings can cause fibrinogen to polymerize into fibrin at a faster rate at the wound, thereby achieving rapid coagulation, and this coagulation mechanism is also independent of the temperature and concentration of coagulation factors. In addition, keratin can also stimulate the migration of human keratinocytes and the expression of type IV and VII collagen. Summary of the invention

[0004] This application proposes to design a recombinant keratin dressing to target various factors of chronic difficult-to-heal wounds, and to regulate various factors of chronic difficult-to-heal wounds from multiple aspects of performance, in order to shorten the wound healing time.

[0005] The technical solution of this application is as follows:

[0006] 1. A recombinant keratin protein, the amino acid sequence of which is shown in the following structure:

[0007] (L)n

[0008] Wherein, the amino acid sequence of L is shown in SEQ ID NO:5,

[0009] n is a positive integer greater than or equal to 1.

[0010] 2. The recombinant keratin according to item 1, wherein n is in the range of 5 to 10.

[0011] 3. The recombinant keratin according to item 1, whose amino acid sequence is shown in SEQ ID NO: 1.

[0012] 4. A gene encoding the recombinant keratin according to any one of items 1 to 3.

[0013] 5. The gene according to item 4, wherein the coding gene sequence of the recombinant keratin is as shown in SEQ ID NO: 2.

[0014] 6. An expression cassette, vector or host bacteria containing the coding gene described in item 4 or 5.

[0015] 7. A method for preparing the recombinant keratin described in any one of items 1 to 3, comprising the following steps: introducing the encoding gene described in item 4 or 5 into an expression host bacterium to obtain a recombinant bacterium, culturing the recombinant bacterium and inducing protein expression, and extracting and purifying the protein.

[0016] 8. A product comprising the recombinant keratin according to any one of items 1 to 3;

[0017] Preferably, the product includes cosmetics, medical devices, medicines or medical materials.

[0018] 9. The product according to item 8, wherein the dosage form of the cosmetic is any one of a dressing, an ointment, a cream, an emulsion, a spray, an aerosol, a cream, an aqueous solution, a gel, an oil, a patch, a film, a mud, a powder, a solution, a film coating and a powder.

[0019] 10. The product according to item 8, wherein the product is a product for repairing chronic wounds,

[0020] The chronic wound surface is derived from one or more of diabetic ulcers, bedsores, venous ulcers, arterial ulcers, pressure ulcers, non-healing postoperative wounds, malignant ulcers, radiation ulcers, and immune ulcers.

[0021] 11. A fiber membrane for regulating diabetic wound surface, comprising keratin, wherein the keratin is the recombinant keratin described in any one of items 1 to 3, the keratin with an amino acid sequence of SEQ ID NO: 3, or the keratin with an amino acid sequence of SEQ ID NO: 4.

[0022] 12. The fiber membrane according to item 11, wherein the fiber membrane further comprises natural polymers and their derivatives and synthetic polymers;

[0023] The natural polymer is selected from one or more of collagen, chitosan, gelatin and silk protein;

[0024] The synthetic polymer is selected from one or more of polycaprolactone, polylactic acid, polylactic acid-co-glycolic acid, polyethylene glycol, polyurethane, polyethylene terephthalate, polystyrene, and polyether amide;

[0025] Preferably, the fiber membrane is prepared by electrospinning a mixed solution consisting of natural polymers and their derivatives, synthetic polymers and keratin;

[0026] Further preferably, the mass percentage of the mixture of the synthetic polymer and the natural polymer and their derivatives to the recombinant keratin is 1:(0.1-10), preferably 8:2.

[0027] 13. Use of the recombinant keratin described in any one of items 1 to 3 in the preparation of a medicine or medical device for wound repair, in the preparation of a medicine or medical device for treating wounds, in the preparation of a product for treating diabetic wounds, and in the preparation of cosmetics for skin barrier repair.

[0028] 14. Use of the recombinant keratin described in any one of items 1 to 3 in the treatment of diabetic wounds.

[0029] 15. A method for treating diabetic wounds, comprising administering to a subject in need thereof the recombinant keratin described in any one of items 1 to 3, the product described in any one of items 8 to 10, or the fiber membrane for regulating diabetic wounds described in any one of items 11 to 12.

[0030] This application has the following beneficial technical effects:

[0031] The present application provides a recombinant keratin RK(84-34)8. When the recombinant keratin is used in a liquid dressing, the liquid dressing can be used to stop bleeding and resist bacteria, promote wound healing, prevent secondary wound damage, etc. It can be used for the preparation of drugs, is simple to prepare, easy to operate, and convenient to produce.

[0032] The present application obtains fused human hair keratin RK(84-34)8 through protein design, whole gene synthesis, vector construction and E. coli expression. In the SD rat diabetic chronic wound model experiment, compared with the blank control group (Control) and the negative control group (PG), the RK(84-34)8 / PG fiber membrane has a significant therapeutic effect on the wound, and the healing effect is better than the human hair keratin RK34 / PG fiber membrane and RK84 / PG fiber membrane before the transformation. The fused human hair keratin of the present application has the function of promoting cell proliferation and migration, promoting wound re-epithelialization, and promoting angiogenesis, and has good application prospects in the field of biomedicine.

[0033] When the keratin of the present application is used for wound repair, the repair function is greatly improved, especially for the repair of chronic wounds such as diabetic foot ulcers, bedsores, venous ulcers, etc. It accelerates the formation of new granulation and epithelial tissue during the granulation proliferation and epithelial formation stages, can quickly promote wound healing, promote wound repair, and has hemostatic and anti-inflammatory functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the result of RK(84-34)8 plasmid transformation.

[0035] Figure 2 This is the SDS-PAGE detection result of the expressed and purified fusion human hair keratin RK(84-34)8.

[0036] Figure 3 Schematic diagram of scanning electron microscopy (SEM) of electrospun nanofiber membrane RK(84-34)8 / PG fused with human hair keratin RK(84-34)8.

[0037] Figure 4 These are pictures of rat skin wounds after treatment with Control, PG, RK34 / PG, RK84 / PG and RK(84-34)8 / PG for 0, 3, 7 and 14 days.

[0038] Figure 5 The wound healing rates of Control, PG, RK34 / PG, RK84 / PG and RK(84-34)8 / PG after treatment at different time points. DETAILED DESCRIPTION

[0039] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the implementation of the present application, but the description is based on the general principles of the specification and is not used to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0040] "Amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide or protein. As used herein, the term "amino acid" includes the following 20 natural or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Val or V). In the case where "X" residues are not defined, these should be defined as "any amino acid". The structures of these 20 natural amino acids are shown in, for example, Stryer et al., BioChemistry, 5th edition, Freeman and Company. Additional amino acids such as selenocysteine ​​and pyrrolysine can also be genetically encoded (Stadtman (1996) "Selenocysteine," Annu Rev Biochem. 65: 83-100 and Ibba et al. "Genetic code: introducing pyrrolysine," Curr Biol. 12 (13): R464-R466). The term "amino acid" also includes non-natural amino acids, modified amino acids (e.g., with modified side chains and / or backbones) and amino acid analogs.

[0041] To further illustrate, an amino acid is generally an organic acid comprising a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group and one or more side chains or groups, or an analog of any of these groups. Exemplary side chains include, for example, sulfhydryl, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphino, phosphine, heterocycle, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising a photosensitive crosslinker, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids containing metals, amino acids containing novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photolabile (photocaged) and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids comprising polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids comprising carbon-linked sugars, redox-active amino acids, amino thioacid-containing amino acids, and amino acids comprising one or more toxic moieties.

[0042] The term "nucleotide", in addition to referring to naturally occurring ribonucleotide or deoxyribonucleotide monomers, should also be understood herein to refer to their related structural variants, including derivatives and analogs, which are functionally equivalent with respect to the specific context in which the nucleotide is used, unless the context clearly indicates otherwise.

[0043] The term "codon optimized", "codon-optimized", "codon-optimized" or "codon usage bias" refers to the practice of selecting codons (i.e., codon usage) in a way that optimizes or customizes expression as desired (i.e., a technique for improving protein expression in an organism by increasing the translation efficiency of a target gene). In other words, codon optimization is a method of adjusting codons to match the abundance of host tRNAs, and has traditionally been used to express heterologous genes. New strategies for optimizing heterologous expression take into account global nucleotide content, such as local mRNA folding, codon pair bias, codon ramp or codon correlation. Codon optimization is possible because the degeneracy of codons is inherent. Degeneracy results because there are more codons than coded amino acids. Therefore, the vast majority of amino acids are encoded by multiple codons, which means that there are multiple tRNAs (with different anti-codon loops) that carry any given amino acid. Therefore, different codons can be used without changing the encoded amino acid sequence. That is, a gene or fragment of a nucleic acid can be mutated / altered (or synthesized de novo) to change the codons used to encode a specific amino acid without changing the amino acid sequence of the polypeptide / protein itself. For example, rare codons can be replaced with more abundant codons while keeping the amino acid sequence unchanged.

[0044] The term "host cell" refers to unicellular prokaryotes and eukaryotic organisms (e.g., bacteria, yeasts, and actinomycetes) and unicellular cells from higher plants or animals when grown in cell culture."Host cell" can be an animal host cell, a plant host cell, a yeast host cell, a fungal host cell, a protozoan host cell, and a prokaryotic host cell.

[0045] Expression: The term "expression" in the present context includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0046] The term "vector" refers to a DNA, usually double-stranded, into which a foreign DNA may have been inserted. The vector may be, for example, of plasmid origin. The vector contains a "replicon" polynucleotide sequence that promotes autonomous replication of the vector in the host cell. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell, which, for example, replicates vector molecules, encodes selectable or screenable markers, or encodes transgenics. The vector is used to transport exogenous or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosome DNA or replicate simultaneously with the host chromosome DNA, and can generate several copies of the vector and its inserted DNA. In addition, the vector may also contain the necessary elements that allow the inserted DNA to be transcribed into mRNA molecules or otherwise cause the inserted DNA to be replicated into multiple copies of RNA. Some expression vectors additionally contain sequence elements near the inserted DNA, which increase the half-life of the expressed mRNA, and / or allow the mRNA to be translated into protein molecules. Therefore, many molecules of mRNA and polypeptides encoded by the inserted DNA can be rapidly synthesized.

[0047] Expression vector: The term "expression vector" in the present context includes a linear or circular DNA molecule, which comprises a segment encoding a polypeptide of the present application and the segment may be operably linked to other segments that enable its transcription.

[0048] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be easily processed with recombinant DNA methods and can express the nucleotide sequence. The selection of the vector usually depends on the compatibility of the vector with the host cell into which the vector is introduced. The vector can be a linear or closed circular plasmid.

[0049] The vector may be an autonomously replicating vector, ie, a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, eg, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.

[0050] The vector may contain any means to ensure its own replication. Alternatively, the vector may be integrated into the genome and replicated together with the chromosome into which it is integrated when introduced into the host cell. Alternatively, a single vector or plasmid may be used, or two or more vectors or plasmids may contain in total all the DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0051] Keratin is a general term for a family of tough proteins present in a variety of structures. It is a protein used as a structural element by many types of animals and is a classic example of fibrous protein. In order to achieve this structural function, keratin molecules are spiral and fibrous, winding around each other to form chains called intermediate filaments. It is believed that this makes keratin difficult to be digested by enzymes at the beginning. In addition, keratin contains a high percentage of sulfur-containing amino acids, mainly cysteine, which form disulfide bonds between individual molecules and help to form a fairly rigid keratin structure. Unfortunately, disulfide bonds also make the digestion and degradation of keratin quite difficult. There are two main types of keratin: α-keratin and β-keratin. α-keratin is mainly found in mammalian hair (including wool), animal horns, finger / toe nails, claws and hoofs. Harder β-keratin is found in reptiles' toenails and scales and claws, their shells (Testudines, such as turtles, sea turtles, water turtles), feathers, beaks, claws of birds, and quills of porcupines. β-keratin is mainly formed in the form of β-sheets, but some β-sheets are also present in α-keratin.

[0052] The present application provides a recombinant keratin protein, the amino acid sequence of which is shown in the following structure:

[0053] (L)n

[0054] Wherein, the amino acid sequence of L is shown in SEQ ID NO:5,

[0055] n is a positive integer greater than or equal to 1.

[0056] In some embodiments of the present application, SEQ ID NO: 5 is

[0057] YEEEVVCRANAENEFVALKKDVDAAFMNKSDLEAGGGGSRTVNALEIELQAQHNLRDSLENTLTESEAHGGGGS

[0058] In some embodiments of the present application, n is in the range of 5 to 10. For example, n is selected from any one of 5, 6, 7, 8, 9, and 10.

[0059] In some embodiments of the present application, when n is 8, its amino acid sequence is as shown in SEQ ID NO:1.

[0060] SEQ ID NO: 1:

[0061] (SEQ ID NO: 1)

[0062] The present application provides a gene encoding the recombinant keratin shown in SEQ ID NO: 1.

[0063] In some embodiments of the present application, the encoding gene sequence of the recombinant keratin is shown as SEQ ID NO:2.

[0064] SEQ ID NO:2:

[0065]

[0066] In some embodiments of the present application, the coding region sequence of the above-mentioned recombinant keratin that has been initially screened is optimized according to the codon usage preference of Escherichia coli in the Escherichia coli codon usage preference data table. On the premise of ensuring that the protein sequence of the recombinant keratin remains unchanged and only utilizing the degeneracy of the codons, the codons that are used less frequently in Escherichia coli and that affect the efficiency of ribosome passage during the translation process are replaced with codons that are used more frequently to obtain a codon-optimized nucleic acid sequence. The obtained sequence is shown in SEQ ID NO: 2 in the sequence listing.

[0067] According to the nucleic acid sequence of the target gene shown, the template gene was obtained through whole gene synthesis and sequencing verification, as shown in SEQ ID NO: 2.

[0068] The present application provides an expression cassette, vector or host bacteria encoding the above gene.

[0069] The present application also provides a vector, which includes the above-mentioned nucleic acid sequence; preferably, the recombinant vector is a prokaryotic cell recombinant vector; further preferably, the prokaryotic cell recombinant vector is any one of pET3a, pET9a, pET14b, pET15b, pET16b, pET20b, pET21a, pET22b, pET23a, pET28a, and pET30a.

[0070] The present application also provides a host bacterium, which contains the above-mentioned vector; preferably, the host bacterium is any one of BL21, BL21(DE3), Rosetta(DE3), Rosetta-gami(DE3)pLysS, Rosetta(DE3)pLysS, BL21(DE3)pLysS, OrigamiB(DE3), and OrigamiB(DE3)pLysS.

[0071] In the present application, the host bacteria can be any bacteria known in the art, as long as the purpose of the present application is achieved.

[0072] The present application provides a method for preparing the above-mentioned recombinant keratin, comprising the following steps: introducing the above-mentioned encoding gene into an expression host bacterium to obtain a recombinant bacterium, culturing the recombinant bacterium and inducing protein expression, and extracting and purifying the protein.

[0073] The present application provides a product comprising the above-mentioned recombinant keratin.

[0074] In some embodiments of the present application, the product includes cosmetics, medical devices, medicines or medical materials.

[0075] In some embodiments of the present application, the dosage form of the cosmetic is any one of a dressing, an ointment, a cream, an emulsion, a spray, an aerosol, a cream, an aqueous solution, a gel, an oil, a patch, a film, a mud, a powder, a solution, a film coating and a powder.

[0076] In some embodiments of the present application, the product is a product for repairing chronic wounds, and the chronic wounds are derived from one or more of diabetic ulcers, bedsores, venous ulcers, arterial ulcers, pressure ulcers, non-healing postoperative wounds, malignant ulcers, radiation ulcers, and immune ulcers.

[0077] Chronic wounds are wounds that cannot heal within the normal healing time (usually 4-6 weeks). These wounds cannot enter the normal healing process smoothly due to various reasons. Common types include:

[0078] 1. Diabetic ulcer

[0079] Diabetic foot ulcer: Due to the neuropathy and poor blood circulation caused by diabetes, it often occurs in the feet and is a common complication in diabetic patients.

[0080] Diabetic skin ulcers: In addition to the feet, diabetes may also cause skin ulcers in other parts of the body.

[0081] 2. Pressure ulcers (bedsores)

[0082] Stage I pressure ulcer: The skin is intact, but redness, swelling, increased temperature, nodules or pain occur.

[0083] Stage II pressure ulcer: Partial loss of skin thickness, presenting as a superficial open ulcer.

[0084] Stage III pressure ulcer: full-thickness skin loss with visible subcutaneous fat tissue but no fascia involvement.

[0085] Stage IV pressure ulcers: involve full thickness loss of muscle, bone, or supporting tissue.

[0086] 3. Venous ulcers

[0087] Venous leg ulcers: Ulcers on the lower limbs caused by poor venous return, usually on the inner side of the calf.

[0088] 4. Arterial ulcer

[0089] Arterial leg ulcers: ulcers caused by insufficient blood supply due to arteriosclerosis or other reasons, commonly found in distal parts such as toes and ankles.

[0090] 5. Pressure ulcers

[0091] Pressure wound: Local tissue ischemic necrosis caused by long-term pressure, common in patients who are bedridden for a long time or have difficulty in moving.

[0092] 6. Non-healing postoperative wounds

[0093] Postoperative wound infection: A wound that fails to heal after surgery due to infection or other reasons.

[0094] Postoperative dehiscence: The surgical incision fails to heal properly and dehiscence occurs.

[0095] 7. Malignant ulcer

[0096] Cancerous ulcer: an ulcer caused by direct erosion of a malignant tumor or secondary infection.

[0097] 8. Radiation ulcer

[0098] Radiation therapy ulcers: Ulcers caused by chronic damage to the skin and soft tissues after radiation therapy.

[0099] 9. Immune ulcer

[0100] Immune-mediated wounds: Ulcers caused by abnormal immune responses in patients with rheumatoid arthritis or systemic lupus erythematosus.

[0101] Chronic wounds have the following characteristics:

[0102] Long-term non-healing: no healing after more than 4-6 weeks;

[0103] Recurrent infection: Recurrent infection is prone to occur, making treatment more difficult;

[0104] Tissue necrosis: Tissue necrosis may occur and debridement is required;

[0105] Pain and discomfort: often accompanied by persistent pain and discomfort, affecting the patient's quality of life;

[0106] Poor blood supply: limited by circulation problems, such as diabetes or arteriosclerosis;

[0107] Chronic inflammation: Chronic inflammation in the wound area exists for a long time.

[0108] In some embodiments of the present application, the product is administered by drip, external application or other available methods.

[0109] The present application provides a fibrous membrane for regulating diabetic wound surface, which comprises keratin, wherein the keratin is the recombinant keratin described in SEQ ID NO: 1, the keratin with an amino acid sequence of SEQ ID NO: 3, or the keratin with an amino acid sequence of SEQ ID NO: 4.

[0110] In some embodiments of the present application, the fiber membrane also includes natural polymers and their derivatives and synthetic polymers; the natural polymers are selected from one or more of collagen, chitosan, gelatin, and silk protein; the synthetic polymers are selected from one or more of polycaprolactone, polylactic acid, polylactic acid-hydroxyacetic acid copolymer, polyethylene glycol, polyurethane, polyethylene terephthalate, polystyrene, and polyether amide.

[0111] In some embodiments of the present application, the fiber membrane is prepared by electrospinning a mixed solution consisting of natural polymers and their derivatives, synthetic polymers and the above-mentioned keratin.

[0112] In some embodiments of the present application, the mass percentage of the mixture of the synthetic polymer and the natural polymer and their derivatives to the recombinant keratin is 1:(0.1-10), preferably 8:2; for example, the mass percentage of the mixture of the synthetic polymer and the natural polymer and their derivatives to the recombinant keratin can be 1:0.1, 1:0.2, 1:0.25, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any range therebetween.

[0113] In some embodiments of the present application, the electrospinning process is as follows:

[0114] (1) Preparation of electrospinning masterbatch

[0115] Precisely weigh polycaprolactone (PCL) and gelatin (Gel) and dissolve them in hexafluoroisopropanol (HFIP). Stir them for 24 hours at room temperature to completely dissolve them. Add gelatin to PCL to increase the hydrophilicity of the fiber. The mass ratio of gelatin to PCL is 1: (0.1-10). Stir them for 24 hours at room temperature to completely dissolve them to obtain PCL / Gel (denoted as PG) electrospinning mother solution. After the PG mother solution is completely dissolved, weigh keratin and add it to the solution. Continue stirring for 24 hours at room temperature to obtain the keratin / PG electrospinning solution.

[0116] (2) Electrospinning process

[0117] Use a sterile syringe to absorb a certain amount of spinning solution and install the spinning needle, fix the syringe on the high-voltage electrospinning injection pump, connect the positive electrode of the high-voltage electrostatic device to the spinning needle, connect the negative electrode to the drum receiver, and wrap a layer of tin foil on the drum receiver to receive the spun fibers, thereby obtaining a fiber membrane for regulating diabetic wounds.

[0118] The present application provides the use of the above-mentioned recombinant keratin in the preparation of a product for treating diabetic wounds.

[0119] The present application provides the use of the above-mentioned recombinant keratin in the preparation of drugs or medical devices for wound repair, in the preparation of drugs or medical devices for treating wounds, in the preparation of products for treating diabetic wound repair, and in the preparation of cosmetics for skin barrier repair.

[0120] The present application provides a method for treating diabetic wounds, which comprises administering the recombinant keratin of the present application, the product for regulating diabetic wounds of the present application, or the fiber membrane for regulating diabetic wounds to a subject in need thereof.

[0121] In this application, the term "subject" generally refers to a cell or an animal, which can be a mammal, such as a human, a non-human primate (ape, gibbon, gorilla, chimpanzee, orangutan, macaque), livestock (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cattle, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents and adult subjects. Subjects also include animal disease models, and other animal models known to those skilled in the art.

[0122] In the present application, "diabetic wound" refers to a chronic wound that occurs in diabetic patients. Chronic wound refers to a wound whose healing is delayed due to poor blood circulation caused by peripheral vascular damage caused by diabetes.

[0123] In the present application, the diabetic wound may be caused by burns, ulcers, injuries, surgical operations, plastic surgery, implantation, childbirth, chronic wounds or dermatitis of the epidermis, dermis or subcutaneous tissue of the skin in a diabetic state. More specifically, it may be caused by one or more than one selected from diabetic foot ulcers, diabetic angiopathy and diabetic foot infection. The composition of the present disclosure can be applied to a patch as a medical device.

[0124] As used herein, the term "chronic wound" refers to a wound that does not heal within thirty days.

[0125] As used herein, "wound repair," "wound healing," or "tissue regeneration" refers to the reconstruction of functional tissue (eg, skin tissue, bone tissue, or mucosa) with minimal or no presence of fibrous tissue that could compromise tissue functionality.

[0126] The terms "healing" or "repair" with respect to wounds refer to the process of repairing a wound by scar formation.

[0127] "Chronic wounds" in this application refer to chronic or difficult-to-heal wounds. Common clinical chronic wounds include traumatic infection, pressure sores, bedsores, diabetic ulcers, venous ulcers, bedsores, chronically infected wounds, nutritional deficiencies, wounds of steroid-treated patients, and radiation wounds. Chronic wounds are prone to hypoxia, ischemia, infection, reduced local growth factors or low immunity at the wound site, resulting in a long healing period or even long-term non-healing.

[0128] In the present application, "chronic wound repair" refers to promoting the healing, cure, repair, tissue regeneration, angiogenesis, hemostasis, etc. of chronic wounds.

[0129] Since chronic wounds cannot undergo the normal healing process of skin wounds, many ulcers last for months or even years without healing. The above seriously affects the quality of life of patients and creates a huge economic burden. If not properly controlled, some wound infections can lead to sepsis and cancer, endangering the patient's life. There are a large number of new dressings, growth factor therapies, cell therapies, etc. for chronic wounds on the market. On the one hand, these technologies are difficult. At the same time, how the above-mentioned cells and growth factors can play a role in the wound for a long time is a difficult problem.

[0130] The recombinant keratin of the present application was shown by SDS-PAGE gel electrophoresis to have high purity of the target protein and can be used for subsequent experimental research.

[0131] The SEM image of the recombinant keratin RK (84-34) 8 electrospun fiber membrane of the present application shows that the surface of the electrospun fiber membrane is smooth, the fiber filaments are continuous, there are no beaded keratin droplets, and the keratin fibers are arranged in a disordered manner. The SEM examination shows that the experiment successfully prepared uniform, continuous and complete RK (84-34) 8 / PG electrospun fiber membrane, RK34 / PG electrospun fiber membrane, and RK84 / PG electrospun fiber membrane.

[0132] The results of the diabetic rat trauma model showed that the wound healing rate of the electrospun fiber membrane fused with human hair keratin RK(84-34)8 / PG was significantly higher than that of the pure fiber and blank control groups, and was better than that of RK84 / PG electrospun fiber membrane and RK34 / PG electrospun fiber membrane. The wound healing rate of the electrospun fiber membrane fused with human hair keratin RK(84-34)8 / PG was 94.31% on the 14th day of treatment of diabetic wounds, which showed significant improvement. The human hair keratin dressing promoted diabetic wound healing significantly and had excellent performance.

[0133] Example

[0134] Bacterial strain: Escherichia coli BL21 (DE3) competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0135] Vector: pET-22(+) plasmid is an E. coli expression vector purchased from Shanghai Biotech Co., Ltd. The promoter of the plasmid is T7 lac, the vector size is 5500 bp, and the vector resistance is Ampicillin. The vector contains the Pel B signal peptide sequence, which can locate the expressed target protein in the cell periplasmic cavity.

[0136] The LB solid medium and LB liquid medium used in the following examples were prepared in the laboratory, and their formulas are as follows:

[0137] LB solid medium: 1.0 g of tryptone, 1.0 g of sodium chloride, 0.5 g of yeast extract, 1.5 g of agar powder, add ddH2O to quantify to 100 mL, and sterilize at 121°C for 20 min.

[0138] LB liquid culture medium: 1.0 g of tryptone, 1.0 g of sodium chloride, 0.5 g of yeast extract, add ddH2O to quantify to 100 mL, sterilize at 121°C for 20 min and set aside.

[0139] Lysis buffer Buffer A: 6.057 g Tris, 8.775 g NaCl, 1.461 g EDTA, 1.5 mL β-mercaptoethanol, add ddH2O to 1 L, adjust pH to 8.0, and store at 4°C.

[0140] Washing solution Buffer B: 6.057g Tris, 8.775g NaCl, 1.461g EDTA, 1.5mL β-mercaptoethanol, 60.5g Urea, 5mL TritonX-100, add ddH2O to quantify to 1L, adjust pH to 8.0, and store at 4℃.

[0141] Denaturation buffer: 6 M guanidine hydrochloride, 1× PBS, 10 mM DTT.

[0142] Renaturation buffer: 50mM Tris HCl, 3mM reduced glutathione, 1mM oxidized glutathione, 5mM EDTA, 0.5M arginine.

[0143] The rats used in the following examples are SD rats purchased from Chongqing Enswell Biotechnology Co., Ltd.

[0144] The experimental reagents not otherwise specified in this application are all conventional reagents in the art, which can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not otherwise specified are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as the molecular cloning experimental manual or the instructions of relevant reagent manufacturers.

[0145] Example 1. Design and expression of fusion of human hair keratin RK(84-34)8

[0146] 1. Protein Design

[0147] The inventors of the present application designed a fusion human hair keratin RK(84-34)8, which mainly comprises one chain, represented by RK(84-34)8. The amino acid sequence of RK(84-34)8 is shown in SEQ ID NO: 1, and its total length is 594 amino acids.

[0148] The amino acid sequence (594aa) of the fused human hair keratin RK(84-34)8 is as follows:

[0149] (SEQ ID NO: 1)

[0150] 2. Expression vector construction

[0151] The amino acid sequence of fused human hair keratin RK (84-34) 8 was translated into the corresponding DNA sequence. The coding gene sequence of fused human hair keratin RK (84-34) 8 is shown in SEQ ID NO: 2, and its full length is 1789 bases. The recognition sequence (GTCGAC) of the restriction endonuclease Sall was added to the 5' end of the coding gene sequence of fused human hair keratin RK (84-34) 8, and the recognition sequence (CTCGAG) of the restriction endonuclease Xhol was added to the 3' end to obtain the target gene sequence of fused human hair keratin RK (84-34) 8. The target gene sequence was commissioned to be fully synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0152] The coding gene sequence (1789 bp) of fused human hair keratin RK (84-34) 8 is as follows:

[0153]

[0154] pET-22(+) plasmid was used as the expression vector. Restriction endonucleases Sall (Thermo Fisher Scientific) and Xhol (Thermo Fisher Scientific) were used to perform double enzyme digestion on pET-22(+) plasmid and the synthesized target gene, respectively. Enzyme digestion system (20 μL): ddH2O 16 μL, 10×Buffer 2 μL, DNA 1 μL, Sall I 0.5 μL, Xhol 0.5 μL. Enzyme digestion conditions: 37°C for 3 hours. Inactivation at 80°C for 20 minutes.

[0155] Recover the pET-22(+) plasmid and target gene after enzyme digestion. Use T4 DNA ligase (New England Biolabs) to connect the target gene to the pET-22(+) vector through ligation reaction. Ligation system (20μL): 10×buffer2μL, T4 DNA ligase0.2μL, pET-22(+)3μL, target gene1μL, ddH2O to 20μL. Ligation conditions: 16℃ overnight.

[0156] The ligation product was transformed into the E. coli DH5α cloning strain using the heat shock method. The transformation method is as follows: Take the E. coli DH5α competent cells out of the -80℃ refrigerator and place them in an ice bath for 5 minutes. After the glycerol of the competent cells melts, add the competent cells to the ligation product, mix them by pipetting 3-4 times with the pipette tip, and place them in an ice bath for 30 minutes. Quickly wipe the water off the tube wall with absorbent paper, place it in a 42℃ heat shock for 90 seconds, and then immediately place it in an ice bath for 2 minutes. Add 800μL of LB liquid culture medium under sterile conditions and culture it at 37℃, 150rpm for 45 minutes. Collect the cells by centrifugation at 8000rpm for 5 minutes, discard part of the supernatant, resuspend the E. coli with the remaining 100μL of supernatant, and then evenly spread it on the LB solid culture medium containing 100μg / mL ampicillin, place it in a 37℃ incubator, and culture it upside down for 10-16 hours. A single clone was picked and inoculated into liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for 10-16 h, the bacterial solution was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing to identify positive clones.

[0157] A plasmid extraction kit (Omega, D6943-01) was used to extract the plasmid of the positive clone with the correct sequence according to the kit instructions to obtain the recombinant plasmid pET-22(+)-MP-KRT82.

[0158] 3. E. coli Transformation

[0159] (1) Take 20 μg of recombinant plasmid and use restriction endonuclease Sac I (Promega) to digest the recombinant plasmid. Enzyme digestion system (20 μL): 16 μL ddH2O, 1 μL recombinant plasmid, 2 μL 10× buffer, 1 μL Sac I. Enzyme digestion conditions: digest at 37°C for 2 h. After inactivation at 65°C for 20 min, use a plasmid extraction kit (Omega, D6943-01) to recover the linearized recombinant plasmid according to the kit instructions.

[0160] (2) Take 15 μL of the recovered linearized recombinant plasmid and mix it with 100 μL of E. coli competent cells in a 1.5 mL EP tube, then transfer it to a 0.2 cm electroporation cup, place it on ice for 10 min, and place it in an electroporator for electroporation. The electroporation conditions are: voltage 1.5 kV, capacitance 25 μF, resistance 200 Ω, and electroporation time 10 ms.

[0161] (3) After the electroporation is completed, remove the electroporation cup and add 1 mL of 1 M sorbitol solution pre-cooled on ice into the electroporation cup. Gently pipette and mix.

[0162] (4) Transfer the liquid in the electroporation transformation cup to a 2 mL EP tube and shake at 30°C for 40 min. Spread all the liquid on LB solid medium and culture at 30°C for 2 days.

[0163] (5) A single colony grown on the LB solid medium was picked up with an inoculation loop and inoculated onto the LB solid medium containing 4.0 mg / mL G418. The culture was incubated at 30° C. overnight, and positive clones were screened to obtain recombinant Escherichia coli pET-22(+)-MP-KRT82.

[0164] 4. Protein Expression and Purification

[0165] (1) Use an inoculation loop to pick up a single colony of the recombinant E. coli and inoculate it into a conical flask containing 30 mL of LB medium. Place it in a constant temperature shaker and culture it overnight at 37°C and 140 rpm until OD600 = 0.6-0.8.

[0166] (2) Transfer the bacterial solution to 1 liter of LB liquid medium for expansion culture and place it in a constant temperature shaker at 37°C and 200 rpm to culture until the OD 600 =0.6-0.8, add IPTG and culture for 4 h to induce protein expression.

[0167] (3) After induction of expression, the cells were collected by centrifugation at 8000 rpm for 10 min at 4°C.

[0168] (4) Add lysis buffer A to resuspend the cell pellet, mix well and use a homogenizer to break the cells. Centrifuge the broken liquid at 4°C, 8000 rpm for 30 min, collect the supernatant, and repeat twice.

[0169] (5) Add Buffer B to the precipitate after centrifugation and mix well. Centrifuge at 4°C, 8000 rpm for 15 min, collect the supernatant, and repeat twice.

[0170] (6) For the remaining inclusion body precipitate, add denaturing buffer to dissolve it, centrifuge at 8000 rpm at 4°C for 30 min, and collect the supernatant.

[0171] (7) The supernatants from all the above steps were purified using a nickel column.

[0172] (8) The purified protein solution was dialyzed against renaturation buffer overnight. After renaturation, the protein solution was dialyzed against 50 mM Tris HCl and centrifuged at 4°C, 8000 rpm for 15 min to remove protein aggregates.

[0173] (9) Arginine was added to the final solution to adjust the protein solubility.

[0174] 5. SDS-PAGE protein detection

[0175] Prepare SDS-PAGE protein electrophoresis gel, wherein the concentration of separation gel is 10% and the concentration of concentration gel is 5%. The formula is as follows:

[0176] Separation gel 10% (10 mL): 30% acrylamide solution 3.3 mL, ddH2O 4 mL, 1.5 M Tris (pH 8.8) 2.5 mL, 10% SDS 0.1 mL, 10% ammonium persulfate solution 0.1 mL, TEMED 0.004 mL.

[0177] Concentrating gel 5% (5 mL): 30% acrylamide solution 0.83 mL, ddH2O 3.4 mL, 1.5 M Tris (pH 6.8) 0.63 mL, 10% SDS 0.05 mL, 10% ammonium persulfate solution 0.05 mL, TEMED 0.005 mL.

[0178] Sample preparation: Take 20 μL of purified protein solution, add Loading Buffer, shake and mix, place in a metal bath at 100 degrees Celsius for 10 minutes to denature the protein, centrifuge after denaturation, and take 10 μL for sample loading. Electrophoresis parameters: constant voltage 70V, 120V after entering the separation gel.

[0179] SDS-PAGE results are as follows Figure 3As shown, the molecular weight of the protein fused to human hair keratin RK(84-34)8 is about 65 kDa, which is consistent with the expected protein molecular weight.

[0180] 6. Preparation of recombinant human hair keratin electrospun fiber membrane

[0181] (1) Preparation of electrospinning masterbatch

[0182] Polycaprolactone (PCL) and gelatin (Gel) were accurately weighed and dissolved in hexafluoroisopropanol (HFIP), and the mixture was sealed and stirred for 24 hours at room temperature to completely dissolve. The mass fraction of PCL was 12wt%, and gelatin was added to PCL to increase the hydrophilicity of the fiber. The mass ratio of gelatin to PCL was 1:9. The mixture was sealed and stirred for 24 hours at room temperature until it was completely dissolved to obtain PCL / Gel (denoted as PG) electrospinning mother solution. After the PG mother solution was completely dissolved, RK(84-34)8 keratin was weighed and added to the solution. The mixture was stirred for 24 hours at room temperature to obtain RK(84-34)8 / PG electrospinning solution, in which the mass ratio of keratin to PCL was 2:8.

[0183] (2) Electrospinning process

[0184] Use a 10mL sterile syringe to draw 5mL of spinning solution and install the spinning needle of a 21G nozzle, fix the syringe on the high-voltage electrospinning injection pump, connect the positive electrode of the high-voltage electrostatic device to the spinning needle, connect the negative electrode to the roller receiver, and wrap a layer of tin foil on the roller receiver to receive the spun fibers to obtain RK(84-34)8 / PG fiber membrane.

[0185] Wherein RK34 / PG represents the fiber membrane formed by RK34 keratin and PG fiber membrane, that is, RK(84-34)8 in the above method is replaced by RK34 keratin;

[0186] RK84 / PG represents the fiber membrane formed by RK84 keratin and PG fiber membrane, that is, RK(84-34)8 in the above method is replaced by RK84 keratin.

[0187] The amino acid sequence of RK34 is SEQ ID NO:3:

[0188] MSYSCCLPSLGCRTSCSSRPCVPPSCHGYTLPGACNIPANVSNCNWFCEGS

[0189] FNGSEKETMQFLNDRLASYLEKVRQLERDNAELEKLIQERSQQQEPLLCP

[0190] SYQSYFKTIEELQQKILCAKAENARLVVNIDNAKLASDDFRSKYQTEQSL

[0191] RLLVESDINSIRRILDELTLCKSDLESQVESLREELICLKKNHEEEVNTLRS

[0192] QLGDRLNVEVDTAPTVDLNQVLNETRSQYEALVEINRREVEQWFATQTE

[0193] ELNKQVVSSSEQLQSCQAEIIELRRTVNALEIELQAQHNLRDSLENTLTESE

[0194] AHYSSQLSQVQSLITNVESQLAEIRCDLERQNQEYQVLLDVRARLECEINTYRSLLESEDCKLPCNPCATTNASGNSCGPCGTSQKGCCN(SEQ ID NO:3)

[0195] The amino acid sequence of RK84 is SEQ ID NO:4:

[0196] MSCRSYRVSSGHRVGNFSSCSAMTPQNLNRFRANSVSCWSGPGFRGLGS

[0197] FGSRSVITFGSYSPRIAAVGSRPIHCGVRFGAGCGMGFGDGRGVGLGPRA

[0198] DSCVGLGFGAGSGIGYGFGGPGFGYRVGGVGVPAAPSITAVTVNKSLLTP

[0199] LNLEIDPNAQRVKKDEKEQIKTLNNKFASFIDKVRFLEQQNKLLETKWSF

[0200] LQEQKCIRSNLEPLFESYITNLRRQLEVLVSDQARLQAERNHLQDVLEGF

[0201] KKKYEEEVVCRANAENEFVALKKDVDAAFMNKSDLEANVDTLTQEIDFL

[0202] KTLYMEEIQLLQSHISETSVIVKMDNSRDLNLDGIIAEVKAQYEEVARRSR

[0203] ADAEAWYQTKYEEMQVTAGQHCDNLRNIRNEINELTRLIQRLKAEIEHA

[0204] KAQRAKLEAAVAEAEQQGEATLSDAKCKLADLECALQQAKQDMARQLC

[0205] EYQELMNAKLGLDIEIATYRRLLEGEESRLCEGVGPVNISVSSSRGGLVCG

[0206] PEPLVAGSTLSRGGVTFSGSSSSVCATSGVLASCGPSLGGARVAPATGDLLST

[0207] GTRSGSMLISEACVPSVPCPLPTQGGFSSCSGGRSSSVRFVSTTTSCRTKY(SEQ ID NO:4)

[0208] Example 2. Experimental study on the repair performance of chronic diabetic wounds

[0209] 1. Establishment of rat type II diabetes model

[0210] The model of type II diabetes rats was established by feeding them with a high-sugar and high-fat diet in the early stage to induce insulin resistance, and then injecting STZ to selectively destroy the pancreatic β cells of rats to cause diabetes in rats. SD male rats were selected for the experiment and fed with 45% high-sugar and high-fat diet for one month to increase their weight to form an obesity model. Fasting for 12-14h before modeling, weighing overnight, and measuring fasting blood sugar. STZ (30mg / kg) dissolved in sodium citrate buffer was injected intraperitoneally, and the blank control group (recorded as Control) was injected with the same dose of citric acid buffer. After injection of STZ, the animals were fed with normal water and food. After 72h of modeling, the blood sugar index of the rats was measured by taking blood from the tail. The index of successful modeling was blood sugar value ≥16.7mmol / L, and the symptoms of "three more and one less" appeared, that is, high blood sugar, high food and water intake, high excretion, weight loss, and yellow hair. The blood sugar of the rat tail vein was measured every other day within one week of injection. If the blood sugar reached the standard, it was considered to be a successful modeling, and subsequent experiments could be carried out. STZ was prepared with citric acid buffer, stored in the dark, and placed on ice. The injection should be completed within 30 minutes after preparation.

[0211] 2. Construction of SD rat skin wound model

[0212] (1) Prepare 8-week-old male SD rats weighing 200-220 g.

[0213] (2) Rats were anesthetized by intraperitoneal injection of 30 mg / kg sodium pentobarbital.

[0214] (3) The rat was fixed in a prone position on an operating table, the skin on the back of the rat was shaved with a razor and disinfected with 75% medical alcohol.

[0215] (4) A 1.5 cm × 1.5 cm mark was made on the back of the rat and a full-thickness excision wound model of the rat back skin was prepared along the marked edge using sterile surgical scissors.

[0216] (5) After disinfecting the wound and surrounding skin with 25% medical iodine, the wound was covered with sterilized RK (84-34) 8 / PG fiber membrane, RK34 / PG fiber membrane, or RK84 / PG fiber membrane.

[0217] (6) Apply 3M medical dressing to the wound to fix the fiber membrane and prevent it from falling off.

[0218] (7) The untreated wounds were used as the control group, and the wounds treated with pure PG fiber membranes were used as the blank fiber group.

[0219] (8) After the experiment, the mice were housed in separate cages to prevent biting and were fed with normal water and food.

[0220] (9) The wound recovery was observed with the naked eye and photographed 0, 3, 7, and 14 days after the injury. The wound area was statistically analyzed using ImageJ software. The wound healing rate was calculated as follows:

[0221] Healing rate (%) = [A0-A t ] / A0×100%

[0222] Where A0 is the initial area of ​​the wound, A t is the wound area after healing after “t” days.

[0223] Experimental Results

[0224] The experimental results are as follows Figure 1 , 2 , 3, 4 and 5 as shown. Figure 1 The results showed that the plasmid was successfully transformed into E. coli BL21, and a single colony containing the target gene was obtained. The expression was further purified and subjected to SDS-PAGE gel electrophoresis. Figure 2 As shown, the target protein is around Maker 75KD and 60KD, which is basically consistent with the predicted molecular weight of the target protein 65KD, and there are no impurity bands after purification, indicating that the target protein has high purity and can be used for subsequent experimental research. Figure 3This is the SEM image of the electrospun fiber membrane fused with human hair keratin RK(84-34)8. The surface of the electrospun fiber membrane is smooth, the fiber filaments are continuous, there are no beaded keratin droplets, and the keratin fibers are arranged in a disordered manner. The SEM examination shows that the experiment successfully prepared a uniform, continuous and complete RK(84-34)8 / PG electrospun fiber membrane.

[0225] Figure 4 as well as Figure 5 The results of the diabetic rat wound model showed that from the 3rd day to the 14th day, the wound healing rate of the fused human hair keratin RK(84-34)8 / PG electrospun fiber membrane was significantly higher than that of the pure fiber and blank control groups, and was better than the recombinant human hair keratin RK84 / PG electrospun fiber membrane and RK34 / PG electrospun fiber membrane. The wound healing rate of the fused human hair keratin RK(84-34)8 / PG electrospun fiber membrane in the treatment of diabetic wounds was 94.31% on the 14th day, which showed significant improvement. The fused human hair keratin dressing has a significant effect on promoting diabetic wound healing and has excellent performance.

[0226] PG represents pure PCL / Gel fiber membrane applied on the skin wound of SD rats as a negative control group;

[0227] Control, SD rats were injected with the same dose of citric acid buffer only, serving as the blank control group;

[0228] RK34 / PG indicates that the fiber membrane formed by RK34 keratin and PG fiber membrane was applied on the skin wound of SD rats;

[0229] RK84 / PG indicates that the fiber membrane formed by RK84 keratin and PG fiber membrane was applied on the skin wound of SD rats;

[0230] RK(84-34)8 / PG indicates that the fiber membrane formed by RK(84-34)8 and PG fiber membrane was applied on the skin wound of SD rats.

Claims

1. A recombinant keratin protein, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A gene encoding the recombinant keratin according to claim 1.

3. The gene according to claim 2, wherein The coding gene sequence of the recombinant keratin is shown in SEQ ID NO:

2.

4. An expression cassette, vector or host bacteria containing the coding gene according to claim 2 or 3.

5. A method for preparing the recombinant keratin according to claim 1, comprising the following steps: introducing the coding gene according to claim 2 or 3 into an expression host bacterium to obtain a recombinant bacterium, culturing the recombinant bacterium and inducing protein expression, and extracting and purifying the protein. A product comprising the recombinant keratin according to claim 1.

7. The product according to claim 6, wherein: The products include cosmetics, medical devices, medicines or medical materials.

8. The product according to claim 7, wherein The dosage form of the cosmetic is any one of a dressing, an ointment, a cream, an emulsion, a spray, an aerosol, a cream, an aqueous solution, a gel, an oil, a patch, a film, a mud, a powder, a solution, a film coating and a granule.

9. The product according to claim 7, wherein: The product is a product for repairing chronic wounds. The chronic wound surface is derived from one or more of diabetic ulcers, bedsores, venous ulcers, arterial ulcers, pressure ulcers, non-healing postoperative wounds, malignant ulcers, radiation ulcers, and immune ulcers.

10. A fiber membrane for repairing diabetic wounds, comprising keratin, wherein the keratin is the recombinant keratin according to claim 1, the keratin having an amino acid sequence of SEQ ID NO: 3, or the keratin having an amino acid sequence of SEQ ID NO:

4.

11. The fiber membrane according to claim 10, wherein: The fiber membrane also includes natural polymers and their derivatives and synthetic polymers.

12. The fiber membrane according to claim 11, wherein: The natural polymer is selected from one or more of collagen, chitosan, gelatin and silk protein; The synthetic polymer is selected from one or more of polycaprolactone, polylactic acid, polylactic acid-glycolic acid copolymer, polyethylene glycol, polyurethane, polyethylene terephthalate, polystyrene and polyether amide.

13. The fiber membrane according to claim 11, wherein The fiber membrane is prepared by electrostatic spinning of a mixed solution consisting of natural polymers and their derivatives, synthetic polymers and keratin.

14. The fiber membrane according to claim 12, wherein: The mass percentage of the mixture of the synthetic polymer, the natural polymer and their derivatives to the recombinant keratin is 1: (0.1-10).

15. The fiber membrane according to claim 14, wherein: The mass percentage of the mixture of the synthetic polymer, the natural polymer and the derivatives thereof to the recombinant keratin is 8:

2.

16. Use of the recombinant keratin according to claim 1 in the preparation of drugs or medical devices for wound repair, in the preparation of drugs or medical devices for treating wounds, in the preparation of products for repairing diabetic wounds, and in the preparation of cosmetics for repairing skin barriers.

17. Use of the recombinant keratin according to claim 1 in preparing a product for treating diabetic wounds.

18. A method for repairing diabetic wounds for non-therapeutic purposes, comprising administering the recombinant keratin of claim 1, the product of any one of claims 6 to 9, or the fiber membrane for repairing diabetic wounds of any one of claims 10 to 15 to a subject in need thereof.

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