Keratin, keratin gel dressing for chronic wound repair

Abstract

The present application provides a keratin and a nucleic acid sequence encoding the same. The present application further provides a keratin gel dressing comprising the keratin of the present application, bFGF and a gel matrix.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to keratin (especially recombinant keratin), a keratin gel dressing comprising the keratin, a preparation method of the keratin gel dressing, and the use of the keratin and the keratin gel dressing in promoting wound healing or repairing chronic wounds. Background Art

[0002] Gel dressings maintain a localized moist environment around wounds, offer excellent breathability, protect wounds from microbial infection, prevent dryness, and offer easy and comfortable dressing changes. They help alleviate wound pain and reduce the workload of medical staff, making them the most commonly used wound dressing. The accelerated aging of the population has led to an increase in age-related diseases, such as diabetic foot, bedsores, and arteriovenous ulcers, spurring a surge in demand for medical dressings. However, functional dressings in the domestic market are largely imported.

[0003] Numerous studies have reported both domestically and internationally that keratin is effective for wound repair. However, the keratin used in these dressings is extracted using traditional chemical extraction methods, and the residual toxic chemical reagents are toxic to human cells. Keratin extracted from wool or chicken and duck feathers, in particular, is animal-derived and presents a potential viral risk. Keratin is a structural protein in mammalian ectoderm cells and is widely present in structures such as hair, skin, and nails. The human body has 54 different types of keratin, with up to 17 different types of keratin found in human hair. Using hair as a raw material, the quality of the raw material cannot be guaranteed between batches, and the extracted material is a mixture of multiple different keratins, not a single substance. Directly using keratin or keratin compositions extracted from nature in this way in the medical field makes it impossible to control the quality of the end product and its safety cannot be guaranteed. Summary of the Invention

[0004] It has been reported that using genetic engineering techniques, expressed through microbial fermentation and purified through separation processes, to produce a single keratin substance can promote fibroblast proliferation and migration, accelerating wound healing. However, due to its unique biological structure and the abundance of cysteine in its amino acid sequence, unoptimized gene sequences, when expressed as inclusion bodies in Escherichia coli, can lead to disulfide bond mismatches. Furthermore, the excessive number of disulfide bonds makes it difficult to effectively renature the protein in later processing steps, hindering the correct structure and biological activity of the natural keratin.

[0005] According to the characteristics of chronic wounds, in addition to the effective ingredients to promote repair, the physical and chemical properties of gel dressings also need to be considered and designed to achieve good film-forming properties, hydrophilic properties, and antibacterial properties.

[0006] The purpose of the present application is to provide keratin, the amino acid sequence of which is shown in SEQ ID No. 2.

[0007] Technical solution of this application:

[0008] 1. A keratin protein, the amino acid sequence of which is shown in SEQ ID NO. 2.

[0009] 2. A nucleic acid sequence encoding the keratin described in item 1.

[0010] 3. The nucleic acid sequence according to item 2, wherein the nucleic acid sequence is shown in SEQ ID NO.3.

[0011] 4. A keratin gel dressing comprising the keratin described in item 1;

[0012] Preferably, the concentration of keratin in the keratin gel dressing is 0.05-0.8 mg / mL.

[0013] 5. The keratin gel dressing according to item 4, further comprising basic fibroblast growth factor (bFGF) and a gel matrix;

[0014] Preferably, the gel matrix is selected from one or both of sodium carboxymethyl cellulose and hydroxyethyl cellulose; more preferably, the gel matrix is sodium carboxymethyl cellulose and hydroxyethyl cellulose.

[0015] 6. The keratin gel dressing according to item 5, wherein the mass proportion of the basic fibroblast growth factor in the keratin gel dressing is 0.01-1 μg / g;

[0016] Preferably, in terms of weight percentage based on the total weight of the keratin gel dressing, the sodium carboxymethyl cellulose is 0.25-1.25 wt %, and the hydroxyethyl cellulose is 0.5-2.5 wt %.

[0017] 7. The keratin gel dressing according to item 4, further comprising a moisturizer, a preservative, and a thickener;

[0018] Preferably,

[0019] The moisturizing agent is selected from one or more of glycerin, propylene glycol, and silanes;

[0020] The preservative is phenoxyethanol;

[0021] The thickener is sorbitol;

[0022] More preferably,

[0023] Calculated by weight percentage of the total weight of the keratin gel dressing, the moisturizing agent accounts for 6-15 wt %, the preservative accounts for 0.3-1.0 wt %, and the thickener accounts for 5-15 wt %.

[0024] 8. A method for preparing a keratin gel dressing,

[0025] Dissolve the gel matrix in water and stir to obtain solution A;

[0026] Dissolve a moisturizer, a thickener, and a preservative in water, mix well, and then add keratin and basic fibroblast growth factor (bFGF) to obtain solution B;

[0027] Solution B was added to solution A, stirred, and then water was added to obtain a keratin gel dressing. The amino acid sequence of the keratin was shown in SEQ ID No. 2.

[0028] 9. Use of the keratin according to item 1, or the keratin gel dressing according to any one of items 4 to 7, or the keratin gel dressing prepared by the method according to claim 8 in the preparation of a medicament for promoting wound healing;

[0029] Preferably, the invention is used in the preparation of a medicament for repairing chronic wounds;

[0030] More preferably, the invention is used in the preparation of a medicament for healing diabetic foot ulcers.

[0031] 10. Use of the keratin described in item 1, or the keratin gel dressing described in any one of items 4 to 7, or the keratin gel dressing prepared by the method described in item 8 in the preparation of a vascular endothelial cell proliferation promoter.

[0032] 11. A drug for promoting wound healing or vascular endothelial cell proliferation, comprising the keratin described in item 1, or the keratin expressed by the nucleic acid sequence described in item 2 or 3, or the keratin gel dressing described in any one of items 4 to 7.

[0033] This application has the following beneficial technical effects:

[0034] Compared with unmodified keratin, the recombinant keratin involved in this application has more advantages and better effects in enhancing cell adhesion, proliferation and migration. For example, the recombinant keratin has better cell proliferation activity and significant effect in promoting cell migration.

[0035] The recombinant keratin of the present application or the keratin gel dressing prepared based on the recombinant keratin can significantly promote wound healing and quickly stop bleeding.

[0036] The keratin gel dressing of the present application contains not only recombinant keratin but also bFGF, which has a good angiogenesis-promoting effect and can be used to prepare wound hemostatic agents, wound healing agents, etc. for the treatment of various wounds, especially diabetic foot ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The SDS-PAGE gel electrophoresis diagram of Example 1;

[0038] Figure 2A The results of the cell proliferation experiment of YL1;

[0039] Figure 2B The results of the cell proliferation experiment of YL2;

[0040] Figure 3 The results of the cell migration experiment of YL1;

[0041] Figure 4 The results of the cell migration experiment of YL2;

[0042] Figure 5 The results of the cell migration experiment of YL3;

[0043] Figure 6 The results of the cell migration experiment of YL4;

[0044] Figure 7 Figure 2 is a diagram of the rat wound repair model of Example 2, Example 4 and the blank control group;

[0045] Figure 8 The figure is a bar graph of the wound healing rates of rats in Example 2, Example 4 and the blank control group;

[0046] Figure 9 Figure 1 is a diagram of the rat wound repair model of Comparative Example 1 and Comparative Example 2;

[0047] Figure 10 The figure is a bar graph of the wound repair and healing rates of rats in Comparative Example 1 and Comparative Example 2. DETAILED DESCRIPTION

[0048] 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 present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the invention. The scope of protection of the present invention shall be as defined in the attached claims.

[0049] "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 naturally occurring 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). Where "X" residues are undefined, these shall be defined as "any amino acid." The structures of these 20 natural amino acids are shown, for example, in 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.

[0050] To illustrate further, an amino acid is generally an organic acid that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or groups, or analogs 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 photosensitive crosslinkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids comprising 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.

[0051] 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.

[0052] The terms "codon optimized," "codon-optimized," "codon-optimized," or "codon usage bias" refer to the practice of selecting codons (i.e., codon usage) in a manner 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 from the presence of more codons than codable 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.

[0053] The term "host cell" refers to unicellular prokaryotic and eukaryotic organisms (e.g., bacteria, yeast, and actinomycetes), as well as unicellular cells from higher plants or animals when grown in cell culture. A "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.

[0054] Expression: The term "expression" in this 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.

[0055] The term "vector" refers to a segment of DNA, typically double-stranded, into which a segment of exogenous DNA may have been inserted. A vector may, for example, be of plasmid origin. A vector contains a "replicon" polynucleotide sequence that promotes autonomous replication of the vector in a host cell. Exogenous DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell and which, for example, replicates the vector molecule, encodes a selectable or screenable marker, or encodes a transgene. Vectors are used to transport exogenous or heterologous DNA into suitable host cells. Once in the host cell, the vector can replicate independently of or simultaneously with the host chromosomal DNA, and can produce several copies of the vector and its inserted DNA. In addition, the vector may also contain 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 also contain sequence elements near the inserted DNA that increase the half-life of the expressed mRNA and / or allow the mRNA to be translated into protein molecules. Thus, many molecules of mRNA and polypeptides encoded by the inserted DNA can be rapidly synthesized.

[0056] Expression vector: The term "expression vector" in this context includes a linear or circular DNA molecule that comprises a segment encoding a polypeptide of the present invention and that is operably linked to other segments that allow its transcription.

[0057] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily processed with recombinant DNA methods and can express the nucleotide sequence. The selection of the vector generally 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.

[0058] 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.

[0059] The vector may comprise any means for ensuring its own replication. Alternatively, the vector may be integrated into the genome and replicated together with the chromosome into which it is integrated upon introduction into the host cell. Alternatively, a single vector or plasmid may be used, or two or more vectors or plasmids may be used that collectively contain the entire DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0060] Keratin is the general term for a family of tough proteins found in a variety of structures. It is a protein used as a structural element by many animal species and is a classic example of a fibrous protein. To achieve this structural function, keratin molecules are helical and fibrous, winding around each other to form chains called intermediate filaments. This is believed to make keratin difficult to digest by enzymes in the first place. Furthermore, keratin contains a high percentage of sulfur-containing amino acids, primarily cysteine, which form disulfide bonds between individual molecules and contribute to the rather rigid keratin structure. Unfortunately, disulfide bonds also make keratin difficult to digest and degrade. There are two main types of keratin: α-keratin and β-keratin. α-keratin is primarily found in mammalian hair (including wool), horns, nails, claws, and hooves. The harder β-keratin is found in the nails, scales, and claws of reptiles, in their shells (Testudinidae, such as turtles, tortoises, and terrapins), in the feathers, beaks, and claws of birds, and in the quills of porcupines. β-keratin is primarily formed as a β-sheet, but some β-sheets are also present in α-keratin.

[0061] Basic Fibroblast Growth Factor (bFGF), also known as Fibroblast Growth Factor-2 (FGF-2), is a mitogen for fibroblasts that was purified by Gospodarowiz et al. in 1974 from extracts of bovine pituitary gland and brain tissue. It is composed of 146 amino acids and is called basic fibroblast growth factor because of its isoelectric point of 9.6. bFGF has a wide range of biological effects, playing a crucial role in angiogenesis, wound healing and tissue repair, tissue regeneration, and the growth and development of neural tissue.

[0062] The present application provides a keratin, in particular a recombinant keratin, the amino acid sequence of the keratin being shown in SEQ ID NO.2.

[0063] The amino acid sequence of keratin is shown in SEQ ID NO.1 (denoted as K31), and the protein has a total length of 416 amino acids. Based on this, the present application further modifies the amino acid sequence shown in SEQ ID NO.1: a large number of cysteines are present at the front end of the original sequence, which easily form a large number of disulfide bonds and mismatches during the E. coli expression and purification process, resulting in reduced efficiency. In addition, the recombinant keratin of the present application also retains the active region of the amino acid sequence shown in SEQ ID NO.1. The modified keratin sequence is shown in SEQ ID NO.2 below, retaining amino acids 57-367 of K31. Among them, SEQ ID NO.1 in the present application is derived from the keratin in CN111202868B.

[0064] SEQ ID NO. 2 is as follows:

[0065] KETMQFLNDRLASYLEKVRQLERDNAELENLIRERSQQQEPLLCPSYQSYFKTIEELQQKILCTKSENARLVVQIDNAKLAADDFRTKYQTELSLRQLVESDINGLRRILDELTLCKSDLEAQVESLKEELLCLKSNHEQEVNTLRCQLGDR LNVEVDAAPTVDLNRVLNETRSQYEALVETNRREVEQWFTTQTEELNKQVVSSSEQLQSYQAEIIELRRTVNALEIELQAQHNLRDSLENTLTESEARYSSQLSQVQSLITNVESQLAEIRSDLERQNQEYQVLLDVRARLECEINTYRSLL ESEDCNL

[0066] The present application also provides a nucleic acid sequence encoding the above keratin, and the nucleic acid sequence is shown as SEQ ID NO.3.

[0067] SEQ ID NO.3 is as follows:

[0068] GAAAAAGAAACCATGCAGTTTCTGAATGATCGTCTGGCGAGCTACCTGGAGAAAGTACGCCAGCTGGAACGCGATAATGCCGAACTGGAAAATCTGATTCGCGAACGCAGCCAGCAGCAGGAACCGCTGCTGTGCCCGAGCTACCAGAGCTATTTTAAAACCATTGAAGAACTGCAGCAGAAAATTCTGTGCACCAAAAGCGAAAACGCGCGCCTGGTTGTACAGATTGATAACGCCAAACTGGCGGCCGATGATTTCCGCACCAAATATCAGACCGAACTGAGCCTGCGCCAGCTGGTGGAAAGCGATATTAACGGTCTGCGCCGTATCCTGGATGAACTGACCCTGTGCAAATCCGATCTGGAAGCGCAGGTGGAAAGCCTGAAAGAAGAACTGCTGTGCCTGAAAAGCAACCATGAACAGGAAGTGAACACCCTGCGCTGCCAGCTGGGCGATCGTCTGAATGTGGAGGTGGATGCGGCCCCGACGGTGGATCTGAACCGCGTGCTGAACGAAACCCGTAGCCAATATGAAGCGCTGGTGGAAACCAACCGTCGTGAAGTGGAACAGTGGTTTACGACTCAGACCGAAGAACTGAATAAACAGGTGGTGAGTAGCTCAGAACAGCTGCAGTCATATCAGGCCGAAATCATTGAACTGCGCCGCACCGTGAACGCGCTGGAAATTGAACTGCAGGCCCAGCACAATCTGCGTGATAGCCTGGAAAATACCCTGACCGAAAGCGAAGCGCGCTATAGCAGCCAGCTGAGCCAGGTACAGAGCCTGATCACCAACGTGGAAAGCCAGCTGGCCGAAATTCGCAGCGATCTGGAACGCCAGAACCAGGAATATCAGGTGCTGCTGGATGTGCGCGCGCGCCTGGAATGCGAAATTAACACCTATCGCAGTCTGCTGGAAAGCGAAGACTGCAACCTG

[0069] In some embodiments of the present application, the coding region sequence of the recombinant keratin protein 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. While 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 translation 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: 3 in the sequence listing.

[0070] According to the nucleic acid sequence of the target gene shown, a template gene was obtained by whole gene synthesis and sequencing verification, as shown in SEQ ID NO: 3.

[0071] The present application also provides a recombinant 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.

[0072] The present application also provides a host cell, which contains the above-mentioned recombinant vector; preferably, the host cell 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.

[0073] The present application also provides a method for preparing the above-mentioned recombinant keratin, which comprises the following steps:

[0074] (a) synthesizing a nucleic acid sequence encoding the recombinant keratin described in SEQ ID NO.2;

[0075] (b) combining the nucleic acid sequence in (a) with a prokaryotic cell recombinant expression vector to obtain a recombinant vector;

[0076] (c) The recombinant vector in (b) is introduced into host cells, followed by culture and induction of expression, and recombinant keratin is obtained through purification.

[0077] The present application provides a keratin gel dressing comprising the above-mentioned keratin.

[0078] In some embodiments of the present application, the concentration of keratin in the keratin gel dressing is 0.05-0.8 mg / mL;

[0079] For example, the concentration of keratin in the keratin gel dressing can be 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8 mg / mL or any range therebetween.

[0080] In some embodiments of the present application, the keratin gel dressing further comprises basic fibroblast growth factor (bFGF) and a gel matrix.

[0081] In some embodiments of the present application, the gel matrix is selected from one or more of sodium carboxymethyl cellulose and hydroxyethyl cellulose.

[0082] In some embodiments of the present application, the gel matrix is sodium carboxymethylcellulose and hydroxyethylcellulose.

[0083] In some embodiments of the present application, the mass percentage of the bFGF in the keratin gel dressing is 0.01-1 μg / g, and the mass percentage of the total weight of the keratin gel dressing is 0.25-1.25 wt %, and the mass percentage of the hydroxyethyl cellulose is 0.5-2.5 wt %;

[0084] For example, the mass proportion of the bFGF in the keratin gel dressing can be 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.07, 0.8, 0.9, 1 μg / g or any range therebetween;

[0085] Calculated as a percentage by weight of the total weight of the keratin gel dressing, the sodium carboxymethyl cellulose is 0.25, 0.5, 0.75, 1, 1.25 wt % or any range therebetween;

[0086] Calculated as a percentage by weight of the total weight of the keratin gel dressing, the hydroxyethyl cellulose is 0.5, 1.0, 1.5, 2.0, 2.5 wt % or any range therebetween.

[0087] In some embodiments of the present application, the keratin gel dressing further comprises a moisturizer, a preservative, and a thickener.

[0088] In some embodiments of the present application, the moisturizer is selected from one or more of glycerin, propylene glycol, and silanes; the preservative is phenoxyethanol; and the thickener is sorbitol.

[0089] In some embodiments of the present application, the moisturizing agent is 6-15 wt %, the preservative is 0.3-1.0 wt %, and the thickener is 5-15 wt %, based on the mass percentage of the total weight of the keratin gel dressing;

[0090] For example, the moisturizing agent is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 wt % or any range therebetween, as a percentage by weight of the total weight of the keratin gel dressing;

[0091] Calculated as a percentage by weight of the total weight of the keratin gel dressing, the preservative is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 wt % or any range therebetween;

[0092] Calculated as a percentage by weight of the total weight of the keratin gel dressing, the thickener is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 wt % or any range therebetween.

[0093] The present application provides a method for preparing the above-mentioned keratin gel dressing, wherein:

[0094] Dissolve the gel matrix in water and stir to obtain solution A;

[0095] Dissolve the moisturizer, thickener, and preservative in water, mix well, and then add keratin and bFGF to obtain solution B;

[0096] Solution B is added to solution A, stirred, and then water is added to obtain a keratin gel dressing. The amino acid sequence of the keratin is shown in Seq ID No. 2.

[0097] The present application provides a use of the above-mentioned keratin, or the above-mentioned keratin gel dressing, or the keratin gel dressing prepared by the above-mentioned method in the preparation of a wound healing medicine.

[0098] In some embodiments of the present application, the above-mentioned keratin, or the above-mentioned keratin gel dressing, or the keratin gel dressing prepared by the above-mentioned method is used in the preparation of a drug for repairing chronic wounds.

[0099] In some embodiments of the present application, the above-mentioned keratin, or the above-mentioned keratin gel dressing, or the keratin gel dressing prepared by the above-mentioned method is used in the preparation of a drug for healing diabetic foot ulcers.

[0100] The present application provides a use of the above-mentioned keratin, or the above-mentioned keratin gel dressing, or the keratin gel dressing prepared by the above-mentioned method in the preparation of a vascular endothelial cell proliferation promoter.

[0101] The present application provides a drug for promoting wound healing or vascular endothelial cell proliferation, comprising the above-mentioned keratin or keratin expressed by the above-mentioned nucleic acid sequence or the above-mentioned keratin gel dressing.

[0102] The drug refers to a "pharmaceutically acceptable" drug or "pharmaceutical composition" and may include a "pharmaceutically acceptable excipient" or not.

[0103] In this application, the term "wound" should be broadly interpreted as and encompasses open wounds and closed wounds, where the skin is torn, cut, punctured, or where trauma causes contusions, or any other surface or other condition or defect on the patient's skin, or wounds that benefit from reduced pressure treatment. Including injuries to the skin, subcutaneous tissue, bone and deep organs or connective tissue that are initiated in any of a variety of ways (e.g., bedsores from prolonged bed rest, wounds induced by trauma, wounds related to surgery, etc.) and have various characteristics. Examples of such wounds include, but are not limited to, abdominal wounds or other large or open wounds that are either caused by surgery, trauma, sternotomy, fasciotomy, or other conditions, dehiscent wounds, acute wounds, chronic wounds, subacute and dehiscent wounds, traumatic wounds, flaps and skin grafts, lacerations, abrasions, contusions, burns, diabetic ulcers, pressure ulcers, stomas, surgical wounds, traumatic ulcers and venous ulcers.

[0104] Examples of wounds contemplated by the present application include, but are not limited to, bruises, abrasions, burns, sunburns, incisions, excisions, surgical wounds, necrotizing fasciitis, ulcers, venous stasis ulcers, diabetic ulcers, bedsores, aphthous ulcers, scars, alopecia areata, dermatitis, allergic contact dermatitis, atopic dermatitis, fragrance dermatitis, diaper dermatitis, dyshidrotic dermatitis, psoriasis, eczema, erythema, warts, anal warts, hemangiomas, cherry angiomas, athlete's foot, atypical moles, basal cell carcinoma, Bateman's purpura, bullous pemphigoid, Candida species, chondrosarcoma, Clark's nevus, cold sores, condyloma, cysts, Darier's disease, dermatofibroma, discoid lupus erythematosus, nummular eczema, atopic eczema, dyshidrosis, hand eczema, polymorphous nodules Erythema nodularis, Fordyce's condition, keloid folliculitis nuchal, folliculitis, granuloma annulare, Grover's disease, heat rash, herpes simplex, herpes zoster (shingles), hidradenitis suppurativa, urticaria, hyperhidrosis, ichthyosis, impetigo, keratosis pilaris, keloids, keratoacanthoma, lichen planus, keratosis lichenoides planus, lichen simplex chronicus, lichen sclerosus, lymphomatoid papulosis, lupus cutis, Lyme's disease, lichen striatus, myxoid cysts, mycosis fungoides, molluscum contagiosum, fetal molluscum, nail fungus, diabetic liponecrosis, nummular dermatitis, onycholysis, onychomycosis, pityriasis lichenoides, pityriasis rosea, pityriasis rubra pilaris, plantar warts, poison ivy, poison oak, dyshidrosis, pseudofolliculitis barbae, pruritus anus, and pityriasis alba.

[0105] Wounds are generally classified into four grades, depending on wound depth: (i) Grade I: wounds limited to the epithelium; (ii) Grade II: wounds extending into the dermis; (iii) Grade III: wounds extending into the subcutaneous tissue; and (iv) Grade IV, also known as full-thickness wounds: wounds in which bone is exposed (e.g., bony pressure points such as the greater trochanter or sacrum).

[0106] As used herein, the term "partial thickness wound" includes grade I-III wounds; examples of partial thickness wounds include burns, pressure sores, venous stasis ulcers, and diabetic foot ulcers.

[0107] As used herein, the term "deep wound" is meant to include grade III and IV wounds.

[0108] In this application, "acute wound" refers to skin that has been damaged as a result of a traumatic abrasion, tear, or superficial injury and that has healed spontaneously without complications through the standard stages of wound healing (hemostasis, inflammation, proliferation, and remodeling).

[0109] As used herein, the term "chronic wound" refers to a wound that does not heal within thirty days.

[0110] As used herein, "wound repair," "wound healing," or "tissue regeneration" refers to the reconstruction of functional tissue (e.g., skin tissue, bone tissue, or mucosa) with minimal or no presence of fibrous tissue that could compromise tissue functionality.

[0111] The terms "healing" or "repair" with respect to wounds refer to the process of repairing a wound by scarring.

[0112] "Chronic wounds" in this application refer to wounds that are chronic or difficult to heal. Common clinically recognized chronic wounds include wound infections, pressure sores, bedsores, diabetic ulcers, venous ulcers, bedsores, chronically infected wounds, wounds caused by nutritional deficiencies, wounds of patients on steroids, and radiation-induced wounds. Chronic wounds are prone to hypoxia, ischemia, infection, decreased local growth factors, or low immunity at the affected site, resulting in a prolonged healing period or even prolonged non-healing.

[0113] In this application, "chronic wound repair" refers to promoting the healing, cure, repair, tissue regeneration, angiogenesis, hemostasis, etc. of chronic wounds.

[0114] Because chronic wounds cannot undergo the normal healing process of skin wounds, many ulcers last for months or even years without healing. This 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 currently a large number of new dressings, growth factor treatments, and cell therapies for chronic wounds on the market. On the one hand, these technologies are difficult, and on the other hand, how to ensure that the above-mentioned cells and growth factors can play a long-term role in the wound is a difficult problem.

[0115] The dressing provided in this application can effectively treat chronic wound repair that is currently difficult to overcome, and provides a beneficial solution for treating such intractable wounds.

[0116] The primary goal of wound care is wound closure. Open skin wounds are a major type of wound and include burns, neuropathic ulcers, pressure ulcers, venous stasis ulcers, and diabetic ulcers.

[0117] Open skin wounds typically heal through a process involving six major steps: (i) inflammation; (ii) fibroblast proliferation; (iii) angiogenesis; (iv) connective tissue formation; (v) epithelialization; and (vi) wound contraction. When these steps, individually or collectively, do not function properly, wound healing is impaired. Many factors can impair wound healing, including malnutrition, infection, pharmacological agents (such as actinomycin and steroids), advanced age, and diabetes [see Hunt and Goodson, Current Surgical Diagnosis & Treatment (Way; Appleton & Lange), pp. 86-98 (1988)]. A common problem with wound healing also occurs after surgical procedures on various parts of the body, where the procedure is successful but the open wound does not heal.

[0118] For example, diabetic foot ulcers (DFUs) are a common and serious complication of diabetes. Due to insufficient blood supply to the wound surface and impaired granulation tissue formation, they are the main factors that contribute to the difficulty in wound healing in diabetic patients. Therefore, promoting wound healing in diabetic foot ulcers is crucial for delaying the progression of diabetic foot ulcers, reducing amputation rates, and improving the quality of life of diabetic patients.

[0119] Vascular disease, neuropathy, and infection are generally considered the three most important risk factors for diabetic foot ulcers. Neuropathy primarily involves the loss of self-protective mechanisms, increased local pressure on the plantar surface, impaired temperature regulation, and dry skin, leading to ulcer formation. Vascular disease is caused by localized vascular occlusion, reduced vascular beds, and thickening of the vascular basement membrane, leading to ischemia in the foot and a lack of oxygen and nutrients in local tissues. Diabetic patients have a weakened immune system, and once an ulcer forms, it is prone to secondary infection, which hinders wound healing.

[0120] Numerous studies have found that promoting the expression of various growth factors in chronic ulcer wounds, such as epidermal growth factor (EGF), fibroblast growth factor (FGE), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF), can promote wound healing at different stages of wound healing. Although topical application of exogenous growth factors has the potential to accelerate DFU healing, due to their short half-life and susceptibility to degradation by excessive matrix metalloproteinase (MMP) activity in the wound, exogenous growth factors alone are generally ineffective. Therefore, it is necessary to identify suitable carriers for exogenous growth factors to fully demonstrate their effects and thereby effectively promote wound healing.

[0121] In this application, keratin-based biomaterials have the ability to synthesize extracellular matrix, promoting cell-cell and cell-matrix interactions. Keratin-based hydrogels, films, and scaffolds have been widely used in wound healing, hemostasis, drug delivery, bone regeneration, and nerve growth. Keratin-based cell binding site sequences can promote cell adhesion and proliferation. Keratin hydrogels have demonstrated good clinical efficacy in treating recessive dystrophic epidermolysis bullosa.

[0122] The applicant has discovered that keratin can promote the migration of keratinocytes and the synthesis of type IV and VII collagen. Based on the excellent properties of keratin, keratin materials have potential advantages in wound healing. In this application, based on the advantages of recombinant keratin compared to keratin extracts in enhancing cell adhesion, proliferation, and migration, as well as the good angiogenesis-promoting effect of bFGF, this application provides better treatment effects for diabetic foot ulcers, provides guidance for the development of keratin gel dressings and diabetic foot ulcer treatment products, and is of great significance to both basic and applied research on keratin.

[0123] The term "pharmaceutical composition" or "medicament" refers to a mixture containing one or more therapeutically active ingredients and a carrier or excipient (such as a pharmaceutically acceptable carrier or excipient conventional in the art).

[0124] The pharmaceutical composition of the present invention can optionally include an effective amount of a variety of conventional components. As non-limiting examples, the pharmaceutical composition can include one or more of the following substances: fillers, diluents, detergents, buffers, preservatives, pH and toxicity regulators, mechanical protectants, chemical protectants, adsorbents, antioxidants, viscosity modifiers, extenders, excipients, astringents, softeners, demulcents, wetting agents, emulsifiers, transdermal delivery accelerators, controlled release agents, dyes or colorants, stabilizers, lubricants, etc. These and other conventional pharmaceutical additives well known to those skilled in the art can be used in the pharmaceutical composition of the present invention according to the properties of the delivery vehicle.

[0125] In this application, "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications, and with a commensurate and reasonable benefit / risk ratio, within the scope of reasonable medical judgment. Drug approval agencies (e.g., EMA, US-FDA) provide guidance and approve pharmaceutically acceptable compounds, materials, compositions, and / or dosage forms.

[0126] The term "pharmaceutically acceptable excipient" is used herein to refer to a pharmaceutically acceptable substance selected from solvents, dispersion media, diluents, dispersants, suspending aids, surfactants, isotonicity agents, thickeners or emulsifiers, preservatives, polymers, peptides, proteins, cells, hyaluronidase, and mixtures thereof.

[0127] The term "dressing" refers to a covering for wounds and encompasses dressings used in conjunction with transdermal delivery of pharmaceutical agents, such as patches, plasters, bandages, and gauze. The term also includes materials in amorphous or liquid form. The term generally encompasses dressings for application to body surfaces, including internal and external tissues.

[0128] Gel dressings are a network of swollen polymers composed of non-water-soluble polymers that contain a large amount of water. They have excellent water absorption and can undergo repeated hydration when in contact with the wound surface. They have the dual functions of providing moisture to the wound surface and absorbing exudate, making them a new type of medical dressing commonly used in clinical practice. The main characteristics of gel dressings include: containing a large amount of water, which helps to increase collagenase activity, accelerate the dissolution of necrotic tissue, and effectively play a role in exogenous debridement; absorbing wound exudate and reducing bacterial growth; providing a closed, moist healing environment for the wound surface, promoting wound healing; being odorless, highly compliant, significantly reducing the pain and frequency of dressing changes, and being easy to use; not adhering to tissues, not harming new granulation tissue, and reducing secondary injury; being colorless and transparent, facilitating dynamic observation of the wound surface; having good breathability; and good biocompatibility.

[0129] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0130] The recombinant keratin provided in this application, after sequence modification, is more excellent in promoting cell proliferation activity and also has a significant effect in promoting cell migration efficacy. The recombinant keratin of this application or the keratin gel dressing prepared based on the recombinant keratin can significantly promote healing and quickly stop bleeding. In addition, because the keratin gel dressing of this application not only contains recombinant keratin but also contains bFGF, it has a good effect in promoting angiogenesis and can be used as a wound hemostatic agent, wound healing agent, etc. in the treatment of various wounds, especially in the treatment of diabetic foot ulcers.

[0131] Example 1

[0132] 1.1 Synthesis and screening of recombinant keratin amino acid sequences

[0133] The amino acid sequence of keratin is shown in SEQ ID NO.1 (denoted as K31), and the protein has a total length of 416 amino acids. In the structural region of K31, the domain formed by the first amino acid has strong hydrophobicity, and the active structure of keratin is mainly the middle α-helical structure. On this basis, the present application further modifies the amino acid sequence shown in SEQ ID NO.1: There are a large number of cysteines at the front end of the original sequence, which easily form a large number of disulfide bonds and mismatches during the E. coli expression and purification process, resulting in reduced efficiency. After modification, the amino acid sequence of keratin of the present application is shown in SEQ ID NO.2 (denoted as K31-modified), and the recombinant keratin of the present application also retains the active region of the amino acid sequence shown in SEQ ID NO.1. The modified keratin sequence is shown in SEQ ID NO.2 below, retaining amino acids 57-367 of K31.

[0134] 1.2 Preparation of recombinant keratin

[0135] 1.2.1 Amplification of target fragment

[0136] 1) Synthesis of target gene

[0137] The coding region sequence of the recombinant keratin protein obtained through the initial screening was optimized according to the codon usage preference of E. coli in the E. coli codon usage preference data table. While maintaining the protein sequence of the recombinant keratin unchanged and utilizing only the degeneracy of the codons, the codons that were used less frequently in E. coli and would affect the efficiency of ribosome passage during translation were replaced with codons used more frequently to obtain a codon-optimized nucleic acid sequence. The obtained sequence is shown as SEQ ID No. 3 in the sequence listing.

[0138] According to the nucleic acid sequence of the target gene shown, a template gene was obtained by whole gene synthesis and sequencing verification, as shown in SEQ ID NO: 3.

[0139] 2) Design primers based on the nucleic acid sequence of the target gene. Using the synthesized template gene as a template, PCR amplification was performed using primers F-KRT31: CCCATATGGAAAATCTGTATTTTCAGGGTGA (SEQ ID No. 4) and R-KRT31: CGGGATCCCAGGTTGCAGTCTTCGCTTTCCAG (SEQ ID No. 5).

[0140] PCR reaction system: 1 μL of 10 μmol / L primer, 1 μL of target gene or linearized pET28a-His-TEV vector gene, 4 μL of dNTP (2.5 mM each), 10× Buffer (containing Mg 2+)5μL, 1μL Pfu DNA Polymerase, and add water to make the total volume 50μL.

[0141] PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 30 s, 30 cycles; final extension at 72°C for 5 min.

[0142] 3) The PCR amplification product was detected by agarose gel electrophoresis. The amplified target fragment (about 1.0 kb) was the same size as the expected fragment, indicating that the target gene fragment and the pET28a-His-TEV vector gene fragment were obtained.

[0143] 1.2.2 Construction of recombinant plasmid

[0144] 1) Add 4 μL of the purified target gene fragment, 6 μL of the pET28a-His-TEV linearized vector, 2 μL of 10X CloneEZ Buffer, 2 μL of CloneEZ Enzyme, and deionized water to 20 μL, mix, and incubate at 22°C for 30 minutes, then keep on ice for 5 minutes.

[0145] 2) Add 100 μL of DH5α or Top10 competent E. coli cells to the above mixture, flick the tube several times, and incubate on ice for 30 minutes. Heat shock the tube in a 42°C water bath for 90 seconds and incubate on ice for 5 minutes. Add 1 mL of SOC medium to the cells and shake gently at 37°C for 1 hour at 200 rpm. Centrifuge at 5000 rpm for 5 minutes to collect the cells and resuspend them in 100 μL of SOC liquid medium.

[0146] 3) Spread the cells evenly on a plate containing antibiotics and incubate overnight at 37°C. Pick three positive clones and inoculate them into 5 mL of LB medium containing 50 μg / mL kanamycin (Kan) and incubate overnight at 37°C, 220 rpm / min.

[0147] 4) Take 3 mL of bacterial solution from each sample to extract plasmid (Tiangen Plasmid Extraction Kit) and send it to Nanjing GenScript for sequencing. The correctly sequenced plasmid is named pET28a-His-TEV-KERATIN.

[0148] 1.2.3 Construction of genetically engineered Escherichia coli

[0149] 1) Thaw BL21(DE3) competent cells on ice. Add 1 μL of the recombinant plasmid to the BL21(DE3) competent cells and gently mix. After standing on ice for 30 minutes, heat shock at 42°C for 60 seconds and quickly return to ice. Then, add 450 μL of room-temperature LB medium to the competent cells and shake at 37°C, 220 rpm, for 1 hour.

[0150] 2) Then, take 100 μL of the bacterial solution from the tube and spread it onto an LB plate containing the antibiotic Kan. Incubate at 37°C overnight. Pick two positive colonies from the plate and inoculate them into 5 mL of LB medium containing 50 μg / mL Kan. Incubate at 37°C, shaking at 220 rpm for approximately 3 hours. When the OD600 reaches 0.6, collect the bacterial solution for seed. This will yield genetically engineered E. coli.

[0151] 1.2.4 Induction of recombinant protein expression

[0152] 1) Take 20 μL of the genetically engineered E. coli cultured above and place it in 200 mL of LB liquid medium containing Kan resistance, and culture it at 37°C and 140 rpm overnight.

[0153] 2) Transfer the overnight bacterial culture to LB liquid medium containing Kan at a 2% inoculum size and incubate at 37°C, 110 rpm, for approximately 3 hours. Measure the OD600 to approximately 0.6. Add 0.5 mM IPTG to the medium and incubate at 16°C for 16 hours. Induction is stopped when the OD600 reaches 2.0-2.5. Centrifuge the culture at 3800 rpm, 4°C, for 10 minutes to collect the pellet.

[0154] 1.2.5 Protein purification by HisFF affinity chromatography

[0155] 1) Resuspend 20 g of the collected bacterial cell pellet in 100 mL of Tris buffer (50 mM Tris (pH 8.0), 500 mM NaCl, and 5% wt. Glycerol). Disrupt the pellet twice with a homogenizer, mix thoroughly, and centrifuge at 4000 rpm for 0.5 h at 4°C. Since the target protein is present in inclusion bodies, collect the pellet for later use.

[0156] 2) Resuspend the collected precipitate in 40 mL of denaturing buffer (50 mM Tris (pH 8.0), 500 mM NaCl, 5% glycerol, 20 mM β-mercaptoethanol, 8 M urea, balance deionized water). Once fully dissolved, centrifuge at 20,000 rpm for 1 hour. Collect the supernatant and transfer it to a 5 mL HisFF affinity column equilibrated with denaturing buffer.

[0157] 3) Rinse the column with 10x denaturation buffer. Then, perform stepwise elution with denaturation buffer containing 10mM, 20mM, 50mM, 100mM, 200mM, 300mM, and 500mM imidazole. Collect the eluted protein and desalt it with dialysate (25mM Tris pH=8.0, 10mM imidazole, 20mM β-mercaptoethanol, 20mM cysteine, balance deionized water) and exchange the buffer.

[0158] 4) Add TEV enzyme to the protein solution obtained in step 3) and perform enzymatic digestion at 25°C for 2 hours to remove the N-terminal HIS tag. Collect the supernatant by centrifugation. Transfer the supernatant to a 5 mL HisFF affinity column (equilibrated with dialysate), rinse with 3x the dialysate volume, and collect the flow-through.

[0159] 5) Desalination was performed by dialyzing with a dialysate (20 mM β-mercaptoethanol, 20 mM cysteine, the remainder being deionized water) for 12 h, and the target protein was obtained by freeze-drying.

[0160] The modified recombinant keratin was designated K31-modified (or YL2), and the unmodified keratin was designated K31-original (or YL1, i.e., the keratin in CN111202868B). To compare the performance of the modified recombinant keratin, human hair keratin (designated YL3) and chicken and duck feather hydrolyzed keratin (designated YL4) were used as comparisons. YL4 is a commercially available hydrolyzed keratin peptide (purchased from Hubei Jianpeptide Biotechnology Co., Ltd.). YL3 is a keratin extracted using the method described in the reference (Development and assessment of kerateine nanoparticles for use as a hemostatic agent, Materials Science and Engineering C 63 (2016) 352-358).

[0161] Example 2 Preparation of gel dressing

[0162] The modified recombinant keratin YL2 of Example 1 was used to prepare a gel dressing.

[0163] Dissolve 1 g of sodium carboxymethyl cellulose and 2 g of hydroxyethyl cellulose in 10 g of water and stir to obtain solution A;

[0164] Dissolve 10 g of glycerol, 10 g of sorbitol, and 0.8 g of phenoxyethanol in water, mix well, and then add 30 mg of recombinant keratin YL2 and 0.05 mg of bFGF to obtain solution B;

[0165] Solution B was added to solution A, and after stirring, water was added to a final volume of 100 mL to obtain a keratin gel dressing.

[0166] Example 3 Preparation of gel dressing

[0167] The modified recombinant keratin YL2 of Example 1 was used to prepare a gel dressing.

[0168] Dissolve 1 g of sodium carboxymethyl cellulose and 2 g of hydroxyethyl cellulose in 10 g of water and stir to obtain solution A;

[0169] Dissolve 10 g of glycerol, 10 g of sorbitol, and 0.8 g of phenoxyethanol in water, mix well, and then add 70 mg of recombinant keratin YL2 and 0.05 mg of bFGF to obtain solution B;

[0170] Solution B was added to solution A, and after stirring, water was added to a final volume of 100 mL to obtain a keratin gel dressing.

[0171] Example 4 Preparation of gel dressing

[0172] The modified recombinant keratin YL2 of Example 1 was used to prepare a gel dressing.

[0173] 30 mg of recombinant keratin was dissolved in water, stirred, and then water was added to a final volume of 100 mL to obtain a keratin gel dressing.

[0174] Comparative Example 1

[0175] Dissolve 1 g of sodium carboxymethyl cellulose and 2 g of hydroxyethyl cellulose in 10 g of water and stir to obtain solution A;

[0176] Dissolve 10 g of glycerol, 10 g of sorbitol, and 0.8 g of phenoxyethanol in water, mix well, and then add 30 mg of unmodified keratin YL1 and 0.05 mg of bFGF to obtain solution B;

[0177] Solution B was added to solution A, and after stirring, water was added to a final volume of 100 mL to obtain a keratin gel dressing.

[0178] Comparative Example 2

[0179] Dissolve 1 g of sodium carboxymethyl cellulose and 2 g of hydroxyethyl cellulose in 10 g of water and stir to obtain solution A;

[0180] Dissolve 10 g of glycerol, 10 g of sorbitol, and 0.8 g of phenoxyethanol in water, mix well, and then add 0.05 mg of bFGF to obtain solution B;

[0181] Solution B was added to solution A, and after stirring, water was added to a final volume of 100 mL to obtain a keratin gel dressing.

[0182] Table 1 Parameter comparison of Examples 2-3 and Comparative Examples 1-3

[0183]

[0184]

[0185] Experimental example

[0186] Experimental Example 1

[0187] 2μg, 4μg and 8μg of recombinant keratin (YL2) were taken for SDS-PAGE gel electrophoresis experiment; 2μg, 4μg and 8μg of unmodified keratin (YL1) were taken for SDS-PAGE gel electrophoresis experiment respectively; the results are as follows Figure 1 As shown. Figure 1 As can be seen, the molecular weight of YL2 is 36 kDa, and the protein purity can reach over 95%. The higher purity of YL2 can reduce the formation of dimers and achieve the desired modification purpose.

[0188] Experimental Example 2 Cell proliferation experiment

[0189] The assay is performed using the CCK-8 assay. CCK-8 is a rapid and efficient test for assessing cell proliferation. The kit contains WST-8, which is reduced by cellular deoxygenases (DHAs) under the action of the electron carrier 1-Methoxy PMS to a highly water-soluble yellow formazan product. The amount of formazan produced is proportional to the number of viable cells. This property allows direct analysis of cell proliferation. The faster the cell proliferation, the darker the color. For the same cell population, the color intensity is linearly correlated with cell number.

[0190] The specific method is as follows: When the fibroblast L929 cells grow to 95%, the cells are digested, centrifuged and collected, and the cell suspension is prepared with serum-containing DMEM medium and the cells are counted. 3 The cells were inoculated at a density of 100 μL / well, and the volume of cell culture medium was 100 μL / well. Two groups, unmodified YL1 and modified YL2, were set up respectively, and the concentration gradients of the two groups were set as follows: 0.005, 0.01, 0.05, 0.1, 0.14, 0.16, and 0.2 mg / mL. After the cultured cells adhered to the wall, the L929 cells were treated according to the set groups and concentrations. The data acquisition time was set to 0h, 24h, and 48h. After the culture time was reached, the medium was changed, and 10 μL CCK-8 reagent and DMEM medium were added to each well, and the incubation continued for 2 hours. The absorbance of each well was detected at 450nm, and the data were statistically analyzed and the cell growth curve was drawn.

[0191] The results are shown in Figure 2. Compared with the unmodified keratin YL1, the modified recombinant keratin YL2 has better cell proliferation activity under the same concentration conditions.

[0192] Experimental Example 3 Cell migration experiment

[0193] This experimental example 3 uses a cell scratch assay. The cell scratch assay is a simple method for measuring cell migration and repair ability. It is similar to an in vitro wound healing model. On a monolayer of adherent cells cultured in an in vitro culture dish or plate, a line is drawn in the central area of cell growth with a micropipette tip or other hard object. The cells in the central area are removed, and then the cells are cultured for the set experimental time (e.g., 24 hours). The cell culture plate is removed and the surrounding cells are observed to see whether they migrate (repair) to the central scratch area to determine the cell growth and migration ability.

[0194] The specific method is as follows: when the fibroblast L929 cells grow to 95%, the cells are digested, centrifuged at 1000 rpm for 5 min and the cells are collected. The cells are then prepared into a cell suspension using serum-containing DMEM complete medium and the cells are counted. 5 Cells were seeded into 6-well plates at 1.5 mL of culture medium per well. After cells adhered to the plate in a 37°C, 5% CO2 incubator, a 200 μL pipette tip was used to create a blank area by slashing a cross vertically against the bottom of the 6-well plate. Floating cells were washed with PBS buffer, and then DMEM basal medium was added. Four groups were set up: modified recombinant keratin YL2, unmodified keratin YL1, human hair-extracted keratin YL3, and commercially available hydrolyzed keratin YL4. Each group was treated with a concentration gradient of 0.05, 0.1, 0.2, 0.4, 0.6, and 0.8 mg / mL. Cells were treated with different concentration gradients in different wells. Images were taken of the same area using a microscope at 0, 24, 48, and 72 hours of treatment. The area of cell migration was analyzed using ImageJ software. The cell healing rate was calculated using the formula to represent the cell migration rate.

[0195] Healing rate = [(area of the initial cell scratch area - area of the cell scratch area at different time points after treatment) / area of the initial cell scratch area] × 100%.

[0196] The results are as follows Figure 3-6 As shown above, the modified K31 keratin, namely YL2, significantly promoted cell migration, outperforming the unmodified keratin proteins YL1, YL3, and YL4. Furthermore, it was found that the effect was particularly significant when the concentration of the recombinant keratin was 0.1 mg / mL.

[0197] Experimental Example 4 Wound Repair

[0198] Select the established rat diabetes model and establish a wound model for diabetic rats: anesthetize the rats, remove hair, and disinfect the skin in the middle of the back with 75% ethanol. Then use surgical scissors to cut out a 1.5×1.5cm full-thickness skin defect wound with a depth of up to the fascia layer, but do not cut the muscle layer and blood vessels. The settings are as follows: a blank control group (no dressing is used), the dressing obtained in Example 4 is subjected to corresponding dressing treatment, the dressing obtained in Example 2 is subjected to corresponding dressing treatment, and the dressings obtained in Comparative Example 1 and Comparative Example 2 are subjected to corresponding dressing treatment. Bandage the wound with medical bandages and gauze to avoid wound infection. Collect wound photos at different time points after surgery (day 0, 3, 6, 9, 11, 14, 18), observe the degree of healing of the wound surface, and use ImageJ software to analyze and calculate the healing rate of each wound. The calculation formula for the healing rate is as follows:

[0199]

[0200] A0 is the initial wound area, A t is the wound area on day “t” after trauma.

[0201] Wound repair results Figure 7-10 As shown in the results, recombinant keratin gel dressing has a significant effect in promoting healing during wound repair.

[0202] From the perspective of wound healing, compared with comparative examples 1-2 and the blank control group, the wound repair rat model of Example 2 and Example 4 is better, and the healing time is short and the healing rate is high. In addition, the wound healing effect of Example 2 is better than that of Example 4.

[0203] Compared with Comparative Example 1, Comparative Example 2 did not add keratin, the wound repair process was much slower than that of Comparative Example 1, the wound was also more serious than that of Comparative Example 1, and the healing rate was lower than that of Comparative Example 1, indicating that keratin plays a certain positive role in wound healing. SEQUENCE LISTING <110> Hymers (Chongqing) Medical Biotechnology Co., Ltd. <120> Keratin, keratin gel dressing for chronic wound repair, and preparation method and application thereof <130> TPF02217 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 416 <212> PRT <213> Artificial sequence <220> <223> Artificial Sequence Description: Synthetic Sequence <400> 1 Met Pro Tyr Asn Phe Cys Leu Pro Ser Leu Ser Cys Arg Thr Ser Cys 1 5 10 15 Ser Ser Arg Pro Cys Val Pro Pro Ser Cys His Ser Cys Thr Leu Pro 20 25 30 Gly Ala Cys Asn Ile Pro Ala Asn Val Ser Asn Cys Asn Trp Phe Cys 35 40 45 Glu Gly Ser Phe Asn Gly Ser Glu Lys Glu Thr Met Gln Phe Leu Asn 50 55 60 Asp Arg Leu Ala Ser Tyr Leu Glu Lys Val Arg Gln Leu Glu Arg Asp 65 70 75 80 Asn Ala Glu Leu Glu Asn Leu Ile Arg Glu Arg Ser Gln Gln Gln Glu 85 90 95 Pro Leu Leu Cys Pro Ser Tyr Gln Ser Tyr Phe Lys Thr Ile Glu Glu 100 105 110 Leu Gln Gln Lys Ile Leu Cys Thr Lys Ser Glu Asn Ala Arg Leu Val 115 120 125 Val Gln Ile Asp Asn Ala Lys Leu Ala Ala Asp Asp Phe Arg Thr Lys 130 135 140 Tyr Gln Thr Glu Leu Ser Leu Arg Gln Leu Val Glu Ser Asp Ile Asn 145 150 155 160 Gly Leu Arg Arg Ile Leu Asp Glu Leu Thr Leu Cys Lys Ser Asp Leu 165 170 175 Glu Ala Gln Val Glu Ser Leu Lys Glu Glu Leu Leu Cys Leu Lys Ser 180 185 190 Asn His Glu Gln Glu Val Asn Thr Leu Arg Cys Gln Leu Gly Asp Arg 195 200 205 Leu Asn Val Glu Val Asp Ala Ala Pro Thr Val Asp Leu Asn Arg Val 210 215 220 Leu Asn Glu Thr Arg Ser Gln Tyr Glu Ala Leu Val Glu Thr Asn Arg 225 230 235 240 Arg Glu Val Glu Gln Trp Phe Thr Thr Gln Thr Glu Glu Leu Asn Lys 245 250 255 Gln Val Val Ser Ser Ser Glu Gln Leu Gln Ser Tyr Gln Ala Glu Ile 260 265 270 Ile Glu Leu Arg Arg Thr Val Asn Ala Leu Glu Ile Glu Leu Gln Ala 275 280 285 Gln His Asn Leu Arg Asp Ser Leu Glu Asn Thr Leu Thr Glu Ser Glu 290 295 300 Ala Arg Tyr Ser Ser Gln Leu Ser Gln Val Gln Ser Leu Ile Thr Asn 305 310 315 320 Val Glu Ser Gln Leu Ala Glu Ile Arg Ser Asp Leu Glu Arg Gln Asn 325 330 335 Gln Glu Tyr Gln Val Leu Leu Asp Val Arg Ala Arg Leu Glu Cys Glu 340 345 350 Ile Asn Thr Tyr Arg Ser Leu Leu Glu Ser Glu Asp Cys Asn Leu Pro 355 360 365 Ser Asn Pro Cys Ala Thr Thr Asn Ala Cys Ser Lys Pro Ile Gly Pro 370 375 380 Cys Leu Ser Asn Pro Cys Thr Ser Cys Val Pro Pro Ala Pro Cys Thr 385 390 395 400 Pro Cys Ala Pro Arg Pro Arg Cys Gly Pro Cys Asn Ser Phe Val Arg 405 410 415 <210> 2 <211> 311 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: artificially synthesized sequence <400> 2 Lys Glu Thr Met Gln Phe Leu Asn Asp Arg Leu Ala Ser Tyr Leu Glu 1 5 10 15 Lys Val Arg Gln Leu Glu Arg Asp Asn Ala Glu Leu Glu Asn Leu Ile 20 25 30 Arg Glu Arg Ser Gln Gln Gln Glu Pro Leu Leu Cys Pro Ser Tyr Gln 35 40 45 Ser Tyr Phe Lys Thr Ile Glu Glu Leu Gln Gln Lys Ile Leu Cys Thr 50 55 60 Lys Ser Glu Asn Ala Arg Leu Val Val Gln Ile Asp Asn Ala Lys Leu 65 70 75 80 Ala Ala Asp Asp Phe Arg Thr Lys Tyr Gln Thr Glu Leu Ser Leu Arg 85 90 95 Gln Leu Val Glu Ser Asp Ile Asn Gly Leu Arg Arg Ile Leu Asp Glu 100 105 110 Leu Thr Leu Cys Lys Ser Asp Leu Glu Ala Gln Val Glu Ser Leu Lys 115 120 125 Glu Glu Leu Leu Cys Leu Lys Ser Asn His Glu Gln Glu Val Asn Thr 130 135 140 Leu Arg Cys Gln Leu Gly Asp Arg Leu Asn Val Glu Val Asp Ala Ala 145 150 155 160 Pro Thr Val Asp Leu Asn Arg Val Leu Asn Glu Thr Arg Ser Gln Tyr 165 170 175 Glu Ala Leu Val Glu Thr Asn Arg Arg Glu Val Glu Gln Trp Phe Thr 180 185 190 Thr Gln Thr Glu Glu Leu Asn Lys Gln Val Val Ser Ser Ser Glu Gln 195 200 205 Leu Gln Ser Tyr Gln Ala Glu Ile Ile Glu Leu Arg Arg Thr Val Asn 210 215 220 Ala Leu Glu Ile Glu Leu Gln Ala Gln His Asn Leu Arg Asp Ser Leu 225 230 235 240 Glu Asn Thr Leu Thr Glu Ser Glu Ala Arg Tyr Ser Ser Gln Leu Ser 245 250 255 Gln Val Gln Ser Leu Ile Thr Asn Val Glu Ser Gln Leu Ala Glu Ile 260 265 270 Arg Ser Asp Leu Glu Arg Gln Asn Gln Glu Tyr Gln Val Leu Leu Asp 275 280 285 Val Arg Ala Arg Leu Glu Cys Glu Ile Asn Thr Tyr Arg Ser Leu Leu 290 295 300 Glu Ser Glu Asp Cys Asn Leu 305 310 <210> 3 <211> 936 <212> Ms <213> artificial sequence <220> <223> Artificial sequence description: Artificial synthesis sequence <400> 3 gaaaaagaaa ccatgcagtt tctgaatgat cgtctggcga gctacctgga gaaagtacgc 120. gcgataatgc gcgataatgc cgaactgga aatctgattc gcgaacgcag ccagcagcag gaccgctgc tgtgcccgag ctaccagagc tattttaaaa ccattgaaga actgcagcag aaaattctgt gcaccaaaag cgaaaacgcg cgcctggttg tacagattga taacgccaaa ctggcggccg atgatttccg caccaatat cagaccgaac tgagcctgcg ccagctggtg gaaagcgata ttaacggtct gcgccgtatc ctggatgaac tgaccctgtg caaatccgat ctggaagcgc aggtggaaag cctgaaagaa gaactgctgt gcctgaaaag caccatgaa caggaagtga acaccctgcg ctgccagctg ggcgatcgtc tgaatgtgga ggtggatgcg gccccgacgg tggatctgaa ccgcgtgctg aacgaaaccc gtagccaata tgaagcgctg gtggaaacca accgtcgtga agtggaacag tggtttacga ctcagaccga agaactgaat aaacaggtgg tgagtagctc agaacagctg cagtcatatc aggccgaaat cattgaactg cgccgcaccg tgaacgcgct ggaattgaa ctgcaggccc agcacaatct gcgtgatagc ctggaaaata ccctgaccga aagcgaagcg cgctatagca gccagctgag ccaggtacag 780 agcctgatca ccaacgtgga aagccagctg gccgaaattc gcagcgatct ggaacgccag 840 aaccaggaat atcaggtgct gctggatgtg cgcgcgcgcc tggaatgcga aattaacacc 900 tatcgcagtc tgctggaaag cgaagactgc aacctg 936 <210> 4 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: artificially synthesized sequence <400> 4 cccatatggaaaatctgtattttcagggtg a 31 <210> 5 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: artificially synthesized sequence <400> 5 cgggatccca ggttgcagtc ttcgctttcc ag 32

Claims

1. Use of keratin having an amino acid sequence as shown in SEQ ID NO. 2 in the preparation of a drug for promoting the healing of chronic wounds.

2. Use of keratin having an amino acid sequence as shown in SEQ ID NO. 2 in the preparation of a drug for promoting the healing of chronic wounds caused by diabetic foot ulcers.

3. Use of a keratin gel dressing in the preparation of a medicament for promoting wound healing, the keratin gel dressing comprising keratin having an amino acid sequence as shown in SEQ ID NO. 2, basic fibroblast growth factor (bFGF), and a gel matrix, wherein the concentration of keratin in the keratin gel dressing is 0.05-0.8 mg / mL; The gel matrix is sodium carboxymethyl cellulose and hydroxyethyl cellulose; The mass percentage of the basic fibroblast growth factor in the keratin gel dressing is 0.01-1 μg / g; in terms of mass percentage of the total weight of the keratin gel dressing, the sodium carboxymethyl cellulose is 0.25-1.25wt%, and the hydroxyethyl cellulose is 0.5-2.5wt%.

4. The use according to claim 3, characterized in that The keratin gel dressing further comprises a moisturizer, a preservative and a thickener.

5. The use according to claim 4, characterized in that The moisturizing agent is selected from one or more of glycerin, propylene glycol, and silanes; The preservative is phenoxyethanol; The thickener is sorbitol.

6. The use according to claim 4, characterized in that Calculated by weight percentage of the total weight of the keratin gel dressing, the moisturizing agent accounts for 6-15 wt %, the preservative accounts for 0.3-1.0 wt %, and the thickener accounts for 5-15 wt %.

7. The use according to claim 3, characterized in that The application is application in preparing medicine for repairing chronic wounds.

8. The use according to claim 7, characterized in that The application is application in preparing medicine for healing diabetic foot ulcers.

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