CN115181171A

CN115181171A - Keratin and keratin gel dressing for repairing chronic wound surface

Info

Publication number: CN115181171A
Application number: CN202210452908.0A
Authority: CN
Inventors: 查国栋
Original assignee: Haimosi Chongqing Medical Biotechnology Co ltd
Current assignee: Haimosi Chongqing Medical Biotechnology Co ltd
Priority date: 2022-04-27
Filing date: 2022-04-27
Publication date: 2022-10-14

Abstract

The present application provides a keratin and nucleic acid sequences encoding the same. The present application further provides a keratin gel dressing comprising a keratin protein of the present application in combination with bFGF and a gel matrix.

Description

Keratin and keratin gel dressing for repairing chronic wound surface

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to keratin (especially recombinant keratin), a keratin gel dressing containing the keratin, a preparation method of the keratin gel dressing, and application of the keratin and the keratin gel dressing in promoting wound healing or chronic wound repair.

Background

The gel dressing can maintain local moist environment around the wound, has good air permeability, protects the wound from microbial infection, prevents the wound from drying, is simple, convenient and comfortable during dressing change, is favorable for relieving the pain of the wound, can also relieve the workload of medical staff, and becomes the most common wound dressing. The aging of the population is accelerated, the number of diseases related to aging is increased, and the requirements on medical dressings are increased rapidly for diabetic feet, bedsores, arteriovenous ulcers and the like. But most functional dressings in domestic market are imported mainly.

A great deal of research reports at home and abroad show that the keratin has ideal effect when being used for wound repair. However, the keratin used in the dressings is extracted by a traditional process in a chemical leaching way, the residue of toxic chemical reagents is toxic to human cells, especially the keratin extracted from wool or chicken and duck feather is of animal origin, and has virus hidden danger. Keratin is a structural protein of mammalian ectodermal cells, widely exists in structures such as hair, skin, nails and the like, and the human body has 54 different kinds of keratin, and the human hair has up to 17 different kinds of keratin. The hair is used as a raw material, the quality of the most original raw material batch cannot be guaranteed, and the hair is extracted to be a mixture of a plurality of different keratin, and is not a single substance. The keratin or keratin composition extracted from nature is directly used in the field of medicine, the quality of a terminal product cannot be controlled, and the safety cannot be guaranteed.

Disclosure of Invention

It has been reported that a single keratin substance is prepared by microbial fermentation expression and a purification and separation process by using a genetic engineering technology, and can promote the proliferation and migration of fibroblasts and accelerate the healing of a wound surface. However, keratin has unique biological structural characteristics, amino acid sequences are rich in a large amount of cysteine, unoptimized gene sequences are expressed in an inclusion body form in escherichia coli, the problem of disulfide bond mismatching exists, the number of disulfide bonds is too large, effective renaturation is not easy to perform through a later process, and the correct structure and biological activity of natural keratin cannot be ensured.

According to the characteristics of chronic wounds, besides the promotion of restoration of functional components, the physical and chemical properties of the gel dressing also need to be considered and designed so as to achieve good film-forming property, hydrophilic property, bacterium-inhibiting property and the like.

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

The technical scheme of the application is as follows:

1. the keratin has an amino acid sequence shown as SEQ ID NO. 2.

2. A nucleic acid sequence encoding the keratin of item 1.

3. The nucleic acid sequence of item 2, which is represented by SEQ ID NO. 3.

4. A keratin gel dressing comprising the keratin of item 1;

preferably, the concentration of the keratin in the keratin gel dressing is from 0.05 to 0.8mg/mL.

5. The keratin gel dressing of claim 4 further comprising a basic fibroblast growth factor (bFGF) and a gel matrix;

preferably, the gel matrix is selected from one or two of sodium carboxymethyl cellulose and hydroxyethyl cellulose; further preferably, the gel matrix is sodium carboxymethyl cellulose and hydroxyethyl cellulose.

6. The keratin gel dressing of item 5, wherein the mass ratio of the basic fibroblast growth factor in the keratin gel dressing is 0.01-1 μ g/g;

preferably, the weight percentage of the sodium carboxymethyl cellulose in the total weight of the keratin gel dressing is 0.25-1.25wt%, and the weight percentage of the hydroxyethyl cellulose in the total weight of the keratin gel dressing is 0.5-2.5wt%.

7. The keratin gel dressing of item 4 further comprising a humectant, a preservative, and a thickener;

preferably, the first and second electrodes are formed of a metal,

the humectant is selected from one or more of glycerin, propylene glycol and silane;

the preservative is phenoxyethanol;

the thickening agent is sorbitol;

it is further preferred that the first and second liquid crystal compositions,

the weight percentage of the humectant accounts for 6-15wt%, the preservative accounts for 0.3-1.0wt%, and the thickening agent accounts for 5-15wt% of the total weight of the keratin gel dressing.

8. A method for preparing a keratin gel dressing,

dissolving the gel matrix in water, and stirring to obtain a solution A;

dissolving a humectant, a thickening agent and a preservative in water, uniformly mixing, and adding keratin and basic fibroblast growth factor (bFGF) to obtain a solution B;

and adding the solution B into the solution A, stirring, and adding water to obtain the keratin gel dressing, wherein the amino acid sequence of the keratin is shown as SEQ ID No. 2.

9. Use of the keratin of claim 1, or the keratin gel dressing of any one of claims 4 to 7, or the keratin gel dressing produced by the method of claim 8, in the manufacture of a medicament for promoting wound healing;

preferably, the application in preparing the medicament for repairing the chronic wound;

further preferably, the application in the preparation of the medicine for curing diabetic foot ulcer.

10. Use of the keratin of claim 1, or the keratin gel dressing of any one of claims 4 to 7, or the keratin gel dressing produced by the method of claim 8 in the production of a vascular endothelial cell proliferation promoter.

11. A medicament for promoting wound healing or vascular endothelial cell proliferation, which comprises the keratin of item 1 or the keratin expressed by the nucleic acid sequence of item 2 or 3 or the keratin gel dressing of any one of items 4 to 7.

The application has the following beneficial technical effects:

compared with unmodified keratin, the recombinant keratin has more advantages and better effects in the aspects of enhancing cell adhesion, proliferation and migration, such as better cell proliferation activity and remarkable cell migration promoting effect of the recombinant keratin.

The recombinant keratin or the keratin gel dressing prepared based on the recombinant keratin can obviously promote wound healing and stop bleeding quickly.

The keratin gel dressing not only contains recombinant keratin, but also has bFGF (basic fibroblast growth factor), has a good effect of promoting angiogenesis, and can be used for preparing wound hemostats, wound healing agents and the like for treating various wounds, particularly diabetic foot ulcers.

Drawings

FIG. 1 is a photograph of SDS-PAGE gel electrophoresis of example 1;

FIG. 2A shows the results of a cell proliferation assay for YL 1;

FIG. 2B shows the results of a cell proliferation assay for YL 2;

FIG. 3 shows the results of a cell migration experiment with YL 1;

FIG. 4 shows the results of a cell migration assay for YL 2;

FIG. 5 shows the results of a cell migration assay on YL 3;

FIG. 6 shows the results of a cell migration experiment with YL 4;

FIG. 7 is a model of wound repair in rats of example 2, example 4 and a blank control group;

FIG. 8 is a bar graph of wound repair healing rates of rats of example 2, example 4 and a blank control group;

FIG. 9 is a model diagram of wound repair in rats of comparative example 1 and comparative example 2;

fig. 10 is a bar graph of the healing rate of the wound repair of rats of comparative example 1 and comparative example 2.

Detailed Description

It should be noted that certain terms are used throughout the description and following claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

"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 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 cases where the "X" residues are undefined, these should be defined as "any amino acid". The structures of these 20 natural amino acids are shown, for example, in Stryer et al, biochemistry, 5 th edition, freeman and Company. Additional amino acids such as Selenocysteine and pyrrolysine can also be genetically encoded (Stadtman (1996) "Selenocysteine," AnnuRevBiochem.65:83-100 and Ibba et al. "Genetic code: introducingpyrrysine," Curr biol.12 (13): R464-R466). The term "amino acid" also includes unnatural amino acids, modified amino acids (e.g., having modified side chains and/or backbones), and amino acid analogs.

To further illustrate, an amino acid is typically 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, mercapto, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halogen, hydrazide, alkenyl, alkynyl, ether, borate, phospho, phosphono, 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 photoactive crosslinkers, metal-binding amino acids, spin-labeled amino acids, fluorescing amino acids, metal-containing amino acids, amino acids containing new functional groups, amino acids that interact covalently or non-covalently 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.

The term "nucleotide", in addition to referring to naturally occurring ribonucleotide or deoxyribonucleotide monomers, is herein understood to refer to related structural variants thereof, 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.

The terms "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 needed (i.e., a technique that improves protein expression in an organism by increasing the translation efficiency of a gene of interest). In other words, codon optimization is a method of adjusting codons to match host tRNA abundance 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 (codon ramp) or codon dependence. Codon optimization is possible because codon degeneracy is inherent. Since there are more codons than can encode an amino acid, resulting in degeneracy. Thus, the vast majority of amino acids are encoded by multiple codons, meaning that there are multiple trnas (with different anti-codon loops) that carry any given amino acid. Thus, different codons can be used without changing the encoded amino acid sequence. That is, a gene or fragment of a nucleic acid may be mutated/altered (or de novo synthesized) to change the codon used to encode a particular amino acid, without altering 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.

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.

Expressing: the term "expression" in this context includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term "vector" refers to a piece of DNA, usually double stranded, into which a foreign piece of DNA may have been inserted. The vector may be of plasmid origin, for example. The vector contains a "replicon" polynucleotide sequence that facilitates 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, which, for example, replicates a vector molecule, encodes a selectable or screenable marker, or encodes a transgene. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector may replicate independently of or simultaneously with the host chromosomal DNA, and several copies of the vector and its inserted DNA may be generated. In addition, the vector may contain the necessary elements to permit transcription of the inserted DNA into an mRNA molecule or to otherwise cause replication of the inserted DNA into multiple copies of RNA. Some expression vectors additionally contain sequence elements near the inserted DNA that increase the half-life of the expressed mRNA and/or allow translation of the mRNA into a protein molecule. Thus, many molecules of mRNA and polypeptide encoded by the inserted DNA can be synthesized rapidly.

Expression vector(s): the term "expression vector" in this context includes a linear or circular DNA molecule comprising a segment encoding a polypeptide of the present invention, which segment may be operably linked to other segments for its transcription.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently treated with recombinant DNA procedures and can express the nucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.

The vector may contain any means for ensuring self-replication (means). Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Alternatively, a single vector or plasmid, or two or more vectors or plasmids which collectively contain the entire DNA to be introduced into the genome of the host cell, may be used, or a transposon may be used.

Keratin is a generic name for a family of tough proteins that exist in a variety of structures. It is a protein used as a structural element by many classes of animals, and is the classic example of fibrin. To achieve this structural function, keratin molecules are helical and fibrous, winding around each other forming chains called intermediate filaments. It is believed that this makes it difficult to digest the keratin initially with the enzyme. In addition, keratin contains a high percentage of sulfur-containing amino acids, mainly cysteines, which form disulfide bonds between individual molecules and contribute to a rather rigid keratin structure. Unfortunately, disulfide bonds also make digestion and degradation of keratin quite difficult. There are two main types of keratin: alpha-keratin and beta-keratin. Alpha-keratin is found mainly in mammalian hair (including wool), horns, nails, claws and hooves. Harder β -keratin is present in the toenails and scales and claws of reptiles, in their shells (of the order of the terrapin, such as turtles, water turtles), in the feathers, beaks, claws of birds, and in the bristles of porcups. Beta-keratin is mainly formed in the form of beta-sheets, but some beta-sheets are also present in alpha-keratin.

Basic Fibroblast Growth Factor (bFGF), also known as Fibroblast Growth Factor (FGF-2), is a mitogen for fibroblasts purified in 1974 from extracts of bovine pituitary and brain tissue by Gospodalozi et al, and is composed of 146 amino acids and is called basic Fibroblast Growth Factor because its isoelectric point is 9.6. bFGF has very wide biological action and plays an important role in the processes of angiogenesis, promotion of wound healing and tissue repair, promotion of tissue regeneration and growth and development of nerve tissues.

The application provides a keratin, in particular a recombinant keratin, the amino acid sequence of which is shown in SEQ ID NO. 2.

The keratin amino acid sequence is shown in SEQ ID NO.1 (marked as K31), and the total length of the protein is 416 amino acids. On the basis, the application further modifies the amino acid sequence shown in SEQ ID NO. 1: the front end of the original sequence has a large amount of cysteine, so that a large amount of disulfide bonds and mismatching are easily formed in the process of escherichia coli expression and purification, and the efficiency is reduced. In addition, the recombinant keratin of the present application simultaneously retains the active region of the amino acid sequence shown in SEQ ID NO. 1. The sequence of the reconstructed keratin is shown as SEQ ID NO.2, and the amino acids at the 56 th position to the 367 th position of K31 are reserved. Among them, SEQ ID NO.1 in the present application is derived from keratin in CN 111202868B.

SEQ ID NO.2 is as follows:

KETMQFLNDRLASYLEKVRQLERDNAELENLIRERSQQQEPLLCPSYQSYFKTIEELQQKILCTKSENARLVVQIDNAKLAADDFRTKYQTELSLRQLVESDINGLRRILDELTLCKSDLEAQVESLKEELLCLKSNHEQEVNTLRCQLGDRLNVEVDAAPTVDLNRVLNETRSQYEALVETNRREVEQWFTTQTEELNKQVVSSSEQLQSYQAEIIELRRTVNALEIELQAQHNLRDSLENTLTESEARYSSQLSQVQSLITNVESQLAEIRSDLERQNQEYQVLLDVRARLECEINTYRSLL ESEDCNL

the application also provides a nucleic acid sequence for coding the keratin, and the nucleic acid sequence is shown as SEQ ID NO. 3.

SEQ ID NO.3 is as follows:

GAAAAAGAAACCATGCAGTTTCTGAATGATCGTCTGGCGAGCTACCTGGAGAAAGTACGCCAGCTGGAACGCGATAATGCCGAACTGGAAAATCTGATTCGCGAACGCAGCCAGCAGCAGGAACCGCTGCTGTGCCCGAGCTACCAGAGCTATTTTAAAACCATTGAAGAACTGCAGCAGAAAATTCTGTGCACCAAAAGCGAAAACGCGCGCCTGGTTGTACAGATTGATAACGCCAAACTGGCGGCCGATGATTTCCGCACCAAATATCAGACCGAACTGAGCCTGCGCCAGCTGGTGGAAAGCGATATTAACGGTCTGCGCCGTATCCTGGATGAACTGACCCTGTGCAAATCCGATCTGGAAGCGCAGGTGGAAAGCCTGAAAGAAGAACTGCTGTGCCTGAAAAGCAACCATGAACAGGAAGTGAACACCCTGCGCTGCCAGCTGGGCGATCGTCTGAATGTGGAGGTGGATGCGGCCCCGACGGTGGATCTGAACCGCGTGCTGAACGAAACCCGTAGCCAATATGAAGCGCTGGTGGAAACCAACCGTCGTGAAGTGGAACAGTGGTTTACGACTCAGACCGAAGAACTGAATAAACAGGTGGTGAGTAGCTCAGAACAGCTGCAGTCATATCAGGCCGAAATCATTGAACTGCGCCGCACCGTGAACGCGCTGGAAATTGAACTGCAGGCCCAGCACAATCTGCGTGATAGCCTGGAAAATACCCTGACCGAAAGCGAAGCGCGCTATAGCAGCCAGCTGAGCCAGGTACAGAGCCTGATCACCAACGTGGAAAGCCAGCTGGCCGAAATTCGCAGCGATCTGGAACGCCAGAACCAGGAATATCAGGTGCTGCTGGATGTGCGCGCGCGCCTGGAATGCGAAATTAACACCTATCGCAGTCTGCTGGAAAGCGAAGACTGCAACCTG

in some embodiments of the present application, the initially screened coding region sequence of the recombinant keratin is optimized according to the codon usage preference of escherichia coli in an escherichia coli codon usage preference data table, and under the premise of ensuring that the protein sequence of the recombinant keratin is not changed and only using the degeneracy of codons, codons which are used in escherichia coli with low frequency and can influence the passing efficiency of a ribosome during translation are replaced by codons with high frequency to obtain a nucleic acid sequence after codon optimization, wherein the obtained sequence is shown as SEQ ID No.3 in a sequence table.

According to the nucleic acid sequence of the target gene, the template gene is obtained through whole-gene synthesis and sequencing verification, and is shown as SEQ ID NO. 3.

The present application also provides a recombinant vector comprising the above-described nucleic acid sequence; preferably, the recombinant vector is a prokaryotic recombinant vector; further preferably, the prokaryotic cell recombinant vector is any one of pET3a, pET9a, pET14b, pET15b, pET16b, pET20b, pET21a, pET22b, pET23a, pET28a and pET30 a.

The present application also provides a host cell comprising the above recombinant vector; preferably, the host cell is any one of BL21, BL21 (DE 3), rosetta-gami (DE 3) pLysS, rosetta (DE 3) pLysS, BL21 (DE 3) pLysS, origamB (DE 3) pLysS.

The application also provides a preparation method of the recombinant keratin, which comprises the following steps:

(a) Synthesizing a nucleic acid sequence encoding the recombinant keratin of SEQ ID No. 2;

(b) Combining the nucleic acid sequence in the step (a) with a prokaryotic cell recombinant expression vector to obtain a recombinant vector;

(c) Introducing the recombinant vector in (b) into a host cell, culturing, inducing expression, and purifying to obtain the recombinant keratin.

The present application provides a keratin gel dressing comprising the keratin as described above.

In some embodiments of the present application, the concentration of the keratin in the keratin gel dressing is from 0.05 to 0.8mg/mL;

for example, the concentration of the keratin in the keratin gel dressing may 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, 0.8mg/mL or any range therebetween.

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

In some embodiments herein, the gel matrix is selected from one or more of sodium carboxymethylcellulose and hydroxyethylcellulose.

In some embodiments herein, the gel matrix is sodium carboxymethyl cellulose and hydroxyethyl cellulose.

In some embodiments of the present application, the bFGF is present in the keratin gel dressing in a mass ratio of 0.01 to 1 μ g/g, the sodium carboxymethylcellulose is present in an amount of 0.25 to 1.25wt%, the hydroxyethylcellulose is present in an amount of 0.5 to 2.5wt%, based on the total weight of the keratin gel dressing;

for example, the mass fraction of bFGF in the keratin gel dressing may be 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.07, 0.8, 0.9, 1 μ g/g or any range therebetween;

the sodium carboxymethyl cellulose accounts for 0.25, 0.5, 0.75, 1, 1.25wt% or any range therebetween in percentage by mass of the total weight of the keratin gel dressing;

the hydroxyethyl cellulose is present in an amount of 0.5, 1.0, 1.5, 2.0, 2.5wt% or any range therebetween, in mass percent based on the total weight of the keratin gel dressing.

In some embodiments of the present application, the keratin gel dressing further comprises a humectant, a preservative, and a thickener.

In some embodiments herein, the humectant is selected from one or more of glycerin, propylene glycol, silanes; the preservative is phenoxyethanol; the thickening agent is sorbitol.

In some embodiments herein, the humectant is 6 to 15wt%, the preservative is 0.3 to 1.0wt%, and the thickener is 5 to 15wt%, based on the weight percentage of the total keratin gel dressing;

for example, the humectant is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15wt% or any range therebetween, as a mass percentage 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, 1.0wt% or any range therebetween, calculated as mass percentage of the total weight of the keratin gel dressing;

the thickening agent is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15wt% or any range therebetween, in mass percent based on the total weight of the keratin gel dressing.

The present application provides a process for the preparation of the above-mentioned keratin gel dressing in which,

dissolving the gel matrix in water, and stirring to obtain a solution A;

dissolving a humectant, a thickening agent and a preservative in water, uniformly mixing, and adding keratin and bFGF to obtain a solution B;

The application provides an application of the keratin or the keratin gel dressing prepared by the method in preparing a medicament for healing wounds.

In some embodiments of the present application, the keratin as described above, or the keratin gel dressing prepared by the method as described above, is used for preparing a medicament for chronic wound repair.

In some embodiments of the present application, the use of the above-described keratin or the above-described keratin gel dressing, or the keratin gel dressing made by the above-described method, in the manufacture of a medicament for the healing of diabetic foot ulcers.

The application provides an application of the keratin, the keratin gel dressing or the keratin gel dressing prepared by the method in preparing a vascular endothelial cell proliferation promoter.

The application provides a medicament for promoting wound healing or promoting vascular endothelial cell proliferation, which comprises the keratin or the keratin gel dressing expressed by the keratin or the nucleic acid sequence.

The drug refers to a "pharmaceutically acceptable" drug or "pharmaceutical composition" that may or may not include a "pharmaceutically acceptable excipient".

In this application, the term "wound" is to be interpreted broadly and covers both open and closed wounds in which the skin is torn, cut, punctured or where the trauma causes a contusion, or any other superficial or other condition or defect on the patient's skin, or otherwise those wounds that benefit from reduced pressure treatment. Including injuries to the skin, subcutaneous tissue, bone and deep organs or connective tissue that originate in any of a variety of ways (e.g., bed sores from prolonged bed rest, trauma-induced wounds, surgical-related wounds, etc.) and have various characteristics. Examples of such wounds include, but are not limited to, abdominal wounds or other large or incised wounds that result from either surgery, trauma, sternotomy, fasciotomy, or other conditions, dehiscent wounds, acute wounds, chronic wounds, subacute and dehiscent wounds, traumatic wounds, flap and skin grafts, lacerations, abrasions, contusions, burns, diabetic ulcers, pressure ulcers, stoma, surgical wounds, traumatic ulcers, venous ulcers, and the like.

Examples of wounds contemplated by the present application include but are not limited to, bruise, abrasion, burn wound, sunburn wound, incision wound, excision wound, surgical wound, necrotizing fasciitis, ulcer, venous stasis ulcer, diabetic ulcer, bedsore, aphthous ulcer, scar, alopecia areata, dermatitis, allergic contact dermatitis, atopic dermatitis, perfume dermatitis, diaper dermatitis, dyshidrosis dermatitis, psoriasis, eczema, erythema, wart, anal wart, hemangioma, cherry hemangioma, tinea pedis, atypical fetal block, basal cell carcinoma, bateman's purpura, bullous pemphigoid, candida, cartilaginous dermatitis Ergothami, clark's nevus, cold sore, condyloma, cyst, darie disease, dermatofibroma, discoid lupus erythematosus, eczema nummularia, atopic eczema, sweat, hand eczema, erythema multiforme nodule, erythema multiforme, erythema, and bruising Fordy's condition, hirsutella keloidis, folliculitis, granulomatosis annulare, grover's disease, heat rash, herpes simplex, herpes zoster (shingles), hidradenitis suppurativa, urticaria, hyperhidrosis, ichthyosis, impetigo, keratosis pilaris, keloids, keratoacanthoma keratoderma, lichen planus-like keratosis, chronic lichen simplex, lichen sclerosus, lymphomatoid papulosis, cutaneous lupus, lyme disease, lichen filiformis, pituitous cysts, mycosis fungoides, contagious molluscum, verruca placenta, nail fungus, diabetic lipoid progressive necrosis, nummulus, onychomycosis, pityriasis lichen, pityriasis rosea, pityriasis rubra peltata, wart, poison ivy, poison oak, pompholyx, pseudofolliculitis barbata, anal itch and white itch.

Depending on wound depth, wounds are generally classified into 4 classes: (I) stage I: wounds limited to the epithelium; (II) stage II: a wound extending into the dermis; (III) stage III: a wound extending into subcutaneous tissue; and (IV) grade IV, also known as full-thickness wounds: wounds in which the bone is exposed (e.g., bone pressure points such as the greater trochanter or sacrum).

The term "partial cutaneous wound" as used herein includes class I-III wounds; examples of partial cortical wounds include burns, pressure sores, venous stasis ulcers and diabetic foot ulcers.

The term "deep wound" as used herein is meant to include both class III and IV wounds.

In this application, "acute wound" means that the skin is damaged as a result of traumatic abrasion, laceration or superficial lesions and automatically heals without complications through the standard stages of wound healing (hemostasis, inflammation, proliferation and remodeling).

The term "chronic wound" as used herein refers to a wound that has not healed within thirty days.

In the present application, "wound repair", "wound healing" or "tissue regeneration" refers to the reconstitution of functional tissue (e.g., skin tissue, bone tissue or mucosa) with minimal or complete absence of fibrous tissue that can compromise tissue functionality.

The term "healing" or "repair" in reference to a wound refers to the process of repairing a wound via scarring.

The term "chronic wound surface" as used herein refers to a chronic or hard-to-heal wound, and clinically common chronic wound surfaces include wound infection, pressure sore, bedsore, diabetic ulcer, venous ulcer, bedsore, chronically infected wound, nutritional deficiency, steroid-treated patient wound, and radioactive wound. The affected face of the chronic wound is easy to have hypoxia, ischemia, infection, reduction of the number of local growth factors or low autoimmunity, so that the healing period is long and even the chronic wound is not healed all the year round.

In the present application, "chronic wound repair" refers to the promotion of healing, repair, tissue regeneration, promotion of angiogenesis, hemostasis, and the like of a chronic wound.

Since the chronic wound surface can not carry out the healing process of normal skin wound, many ulcers can not be healed for a long time, which can last for months or even years. The above seriously affects the quality of life of patients and causes huge economic burden. If the control is not good, some wound infection can cause sepsis and canceration, which endangers the life of the patient. At present, a large number of novel dressings, growth factor treatments, cell therapies and the like aiming at chronic wounds exist on the market, on one hand, the technical difficulties are high, and meanwhile, how the components of the cells and the growth factors can play a role in the wounds for a long time is a difficult problem.

The dressing provided by the application can effectively treat chronic wound repair which is difficult to overcome at present, and provides a beneficial solution for treating refractory wounds.

The primary goal of wound therapy is to allow the wound to close. Open skin wounds are a major wound type and include burns, neuropathic ulcers, pressure sores, venous stasis ulcers and diabetic ulcers.

Open skin wounds typically heal by a process that includes six major links: (i) inflammation; (ii) fibroblast proliferation; (iii) vascular proliferation; (iv) connective tissue generation; (v) epithelialization; (vi) wound contraction. When these links do not function properly, individually or in their entirety, wound healing is affected. Wound healing may be affected by a number of factors, including malnutrition, infection, pharmacological agents (such as actinomycin and steroids), advanced age and diabetes [ see Current Surgical Diagnosis & Treatment of Hunt and Goodson (Way; appleton & Lange), pp.86-98 (1988) ]. The common problem of wound healing also occurs after surgical operation on parts of the body, i.e. successful operation but non-healing of open wounds.

For example, diabetic Foot Ulcer (DFU) is one of the common serious complications of diabetes, and is a major factor causing difficult healing of the wound of a diabetic patient due to the characteristics of insufficient blood supply to the wound, formation of granulation tissue and the like. Therefore, how to promote the healing of the diabetic foot ulcer wound surface has important significance for delaying the development of the diabetic foot ulcer, reducing the amputation rate and improving the life quality of the diabetic patient.

At present, vasculopathy, neuropathy and infection are generally considered as three most important risk factors for the occurrence of diabetic foot ulcer. Neuropathy mainly is the formation of ulcers caused by changes such as autonomic nerves, motor nerves and sensory neuropathy in peripheral nerves, loss of self-protection mechanism, increase of local pressure on soles, thermoregulation disorder, dry skin and the like. The vasculopathy is diabetic foot ulcer caused by partial occlusion of blood vessels, reduction of blood vessel beds, thickening of blood vessel basement membrane and the like, which causes foot ischemia and results in lack of oxygen and nutrient substances in local tissues. The diabetic patients have low immune function and ulcer, and are easy to be secondarily infected so as to hinder wound healing.

Numerous studies have found that the expression of different growth factors such as Epidermal Growth Factor (EGF), fibroblast growth Factor (FGE), insulin-like growth factor (IGF), platelet growth factor (PDGF) etc. at chronic ulcer wounds can produce different effects at different stages of wound healing to promote wound healing. Although topical application of exogenous growth factors is expected to accelerate DFU healing, the use of exogenous growth factors alone is generally not effective due to the short half-life of the growth factors, susceptibility to degradation by excessive Matrix Metalloproteinase (MMPs) activity at the wound, and the like. Therefore, it is necessary to find a suitable carrier for exogenous growth factors to fully reflect the effect thereof, thereby achieving the effect of effectively promoting wound healing.

In the present application, keratin-based biomaterials have the function of synthesizing extracellular matrices, promoting cell-cell and cell-matrix interactions. Hydrogels, films, scaffolds based on keratin have been widely used in wound healing, hemostasis, drug delivery, bone regeneration, nerve growth, and the like. The keratin-based cell binding site sequence can promote cell adhesion and proliferation, and the keratin hydrogel is clinically used to have a good curative effect in treating the hidden malnutrition epidermolysis bullosa.

The applicants have found that keratin promotes keratinocyte migration and collagen type iv, vii synthesis. Keratin materials have potential advantages in wound healing based on the excellent properties of keratin. In the application, based on the advantages of the recombinant keratin in enhancing cell adhesion, proliferation and migration and the good angiogenesis promoting effect of bFGF (basic fibroblast growth factor) compared with a keratin extract, the application provides a better treatment effect on the diabetic foot ulcer, provides guidance for the development of keratin gel dressing and diabetic foot ulcer treatment products, and has important significance for the basic research and application research on keratin.

The term "pharmaceutical composition" or "medicament" refers to a mixture containing one or more therapeutically active ingredients and a carrier or excipient (e.g., a pharmaceutically acceptable carrier or excipient conventional in the art).

An effective amount of various conventional components may optionally be included in the pharmaceutical compositions of the present invention. As non-limiting examples, the pharmaceutical composition may comprise one or more of the following: fillers, diluents, detergents, buffers, preservatives, pH and toxicity adjusting agents, mechanical protectants, chemical protectants, adsorbents, antioxidants, viscosity modifiers, bulking agents, excipients, astringents, emollients, demulcents, humectants, emulsifiers, transdermal delivery promoting agents, controlled release agents, dyes or colorants, stabilizers, lubricants, and the like. These and other conventional pharmaceutical additives known to those skilled in the art may be used in the pharmaceutical compositions of the present invention depending on the nature of the delivery vehicle.

By "pharmaceutically acceptable" herein is meant those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. A pharmaceutical approval authority (e.g., EMA, US-FDA) provides guidance and approves pharmaceutically acceptable compounds, materials, compositions, and/or dosage forms.

The term "pharmaceutically acceptable excipient" is used herein to refer to a pharmaceutically acceptable substance selected from solvents, dispersion media, diluents, dispersions, suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, polymers, peptides, proteins, cells, hyaluronidase, and mixtures thereof.

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

The gel dressing is a reticular polymer swelling body which is composed of non-water-soluble polymers and contains a large amount of moisture, has good water absorption, can generate repeated hydration when the gel is contacted with a wound surface, has double functions of supplying water to the wound surface and absorbing seepage, and is a novel medical dressing which is commonly used clinically at present. The gel dressing is characterized in that: contains a large amount of water, is beneficial to improving the activity of collagenase and accelerating the dissolution of necrotic tissues, and can effectively play the role of exogenous debridement; the exudate of the wound surface is absorbed, and the bacterial reproduction is reduced; a closed wet healing environment is provided for the wound surface, and the wound surface healing is promoted; no peculiar smell, good compliance, capability of obviously reducing dressing change pain and dressing change times and convenient application; the tissue is not adhered, the granulation tissue is not damaged, and the secondary damage is reduced; the wound surface is colorless and transparent, so that dynamic observation of the wound surface is facilitated; good air permeability; the biocompatibility is good.

The materials used in the tests and the test methods are generally and/or specifically described herein, and in the examples below,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

The recombinant keratin provided by the application is more excellent in the aspect of promoting cell proliferation activity after sequence modification, and has a remarkable effect in the aspect of promoting cell migration. The recombinant keratin or the keratin gel dressing prepared based on the recombinant keratin can obviously promote healing and stop bleeding quickly. In addition, the keratin gel dressing contains the recombinant keratin and the bFGF, has good angiogenesis promoting effect, and can be used as a wound hemostatic, a wound healing agent and the like for treating various wounds, particularly diabetic foot ulcer.

Example 1

1.1 Synthesis and screening of recombinant Keratin amino acid sequences

The keratin amino acid sequence is shown in SEQ ID NO.1 (marked as K31), and the total length of the protein is 416 amino acids. In the structural region of K31, a structural domain formed by the first amino acid has strong hydrophobicity, and the active structure of keratin is mainly a middle alpha-helix structure, and on the basis, the application further modifies the amino acid sequence shown in SEQ ID NO. 1: the front end of the original sequence has a large amount of cysteine, so that a large amount of disulfide bonds and mismatching are easily formed in the process of escherichia coli expression and purification, and the efficiency is reduced. After being modified, the keratin amino acid sequence is shown as SEQ ID NO.2 (marked as K31-modified), and the recombinant keratin simultaneously reserves the active region of the amino acid sequence shown as SEQ ID NO. 1. The sequence of the modified keratin is shown as SEQ ID NO.2, and the amino acids 56-367 of K31 are reserved.

1.2 preparation of recombinant Keratin

1.2.1 amplification of fragments of interest

1) Synthesis of the Gene of interest

Optimizing the coding region sequence of the initially screened recombinant keratin according to the codon usage preference of the escherichia coli in an escherichia coli codon usage preference data table, replacing codons which have low use frequency and can influence the passing efficiency of a ribosome in the translation process in the escherichia coli with codons with high use frequency on the premise of ensuring that the protein sequence of the recombinant keratin is not changed and only utilizing the degeneracy of the codons to obtain a nucleic acid sequence after codon optimization, wherein the obtained sequence is shown as SEQ ID No.3 in a sequence table

2) Primers were designed based on the nucleic acid sequence of the target gene. PCR amplification is carried out by taking the synthesized template gene as a template and F-KRT31: CCCATATGGAATCTGTATTTTCAGGGTGA (SEQ ID No. 4) and R-KRT31: CGGGATCCCAGGTTGCAGTCCTCTTCCAG (SEQ ID No. 5) as primers.

And (3) PCR reaction system: mu.L of 10. Mu. Mol/L primer 1. Mu.L, 1. Mu.L of the target gene or linearized pET28a-His-TEV vector gene, 4. Mu.L of dNTPs (2.5 mM each), 10 XBuffer (containing Mg) ²⁺ ) mu.L of Pfu DNA Polymerase was added with water to make up a total volume of 50. Mu.L.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 30s, and 30 cycles; finally, extension is carried out for 5min at 72 ℃.

3) And (3) carrying out agarose gel electrophoresis detection on the PCR amplification product. The size of the target fragment (about 1.0 kb) obtained by amplification is the same as that of the expected fragment, and the target gene fragment and the pET28a-His-TEV vector gene fragment are obtained.

1.2.2 construction of recombinant plasmids

1) mu.L of the purified gene fragment of interest and 6. Mu.L of pET28a-His-TEV linearized vector, 10XCloneZ Buffer, 2. Mu.L CloneeZ Enzyme, 2. Mu.L deionized water to make up 20. Mu.L were mixed, kept at 22 ℃ for 30 minutes, and then kept on ice for 5 minutes.

2) Adding 100 mu L of DH5 alpha or Top10 Escherichia coli competent cells into the mixed solution, gently flicking, and incubating on ice for 30 min; heat shock in 42 ℃ water bath for 90 seconds, and incubation on ice for 5 minutes; adding 1mL of SOC culture medium into the cells, and slightly shaking for 1 hour at 37 ℃ at the rotating speed of 200rpm; the cells were collected by centrifugation at 5000rpm for 5 minutes, and suspended in 100. Mu.L of SOC liquid medium.

3) The cells were spread evenly on antibiotic-containing plates and cultured overnight at 37 ℃. Each of 3 positive clones was inoculated in 5mL of LB medium containing 50. Mu.g/mL of kanamycin (Kan) antibiotic and cultured overnight at 37 ℃ and 220 rpm.

4) 3mL of extracted plasmid (Tiangen plasmid extraction kit) is taken from the bacterial liquid of each sample, sent to Nanjing Kingsler for sequencing, and named as pET28a-His-TEV-KERATIN plasmid with correct sequencing.

1.2.3 construction of genetically engineered Escherichia coli

1) BL21 (DE 3) competent cells were dissolved in ice, and 1. Mu.L of the recombinant plasmid was added to BL21 (DE 3) competent cells and gently mixed. After standing in ice for 30min, it was quickly returned to ice by heat shock at 42 ℃ for 60 s. Then 450. Mu.L of room temperature LB medium was added to the competent cells and shaken at 220rpm for 1 hour at 37 ℃ on a shaker.

2) Then, 100. Mu.L of the cell suspension was applied to an LB plate containing Kan antibiotic and cultured overnight at 37 ℃.2 positive clones were picked from the plate and inoculated in 5mL LB medium containing 50. Mu.g/mL Kan antibiotic, cultured at 37 ℃ for about 3h with shaking at 220rpm, OD600=0.6, and the culture was collected and preserved. Thus obtaining the escherichia coli genetic engineering bacteria.

1.2.4 Induction of recombinant protein expression

1) 20 μ L of the above-mentioned Escherichia coli genetic engineering bacteria were cultured overnight at 37 ℃ and 140rpm in 200mL of LB liquid medium containing Kan resistance.

2) The overnight cultured bacterial culture was inoculated into LB liquid medium containing Kan at 2%, cultured at 37 ℃ for about 3 hours at 110rpm, measured at OD600= about 0.6, 0.5mM IPTG inducer was added to the culture medium, induced at 16 ℃ for 16 hours, measured at OD600=2.0-2.5, and the induction was stopped. The bacterial solution was centrifuged at 3800rpm at 4 ℃ for 10min, and the precipitated cells were collected.

1.2.5HisFF affinity chromatography purification of proteins

1) 20g of the precipitated bacterial cells collected above were taken and resuspended in 100mM Tris buffer containing 50mM Tris (pH 8.0), 500mM NaCl,5% by weight of Glycerol. Crushing for 2 times by a homogenizer, mixing uniformly, centrifuging at 4000rpm at 4 ℃ for 0.5h, and collecting the precipitate for later use because the target protein exists in the inclusion body.

2) The collected pellet was resuspended and solubilized with 40mL denatured buffer (50 mM Tris (pH 8.0), 500mM NaCl,5% glycerol, 20mM β -mercaptoethanol, 8M urea, balance deionized water). After sufficient dissolution, the supernatant was centrifuged at 20000rpm for 1h and transferred to a 5mL HisFF affinity column (equilibrated with denaturing Buffer).

3) The column was washed with 10-fold denaturing buffer. Then, stepwise elution was performed using denatured buffers containing 10mM, 20mM, 50mM, 100mM, 200mM, 300mM, and 500mM imidazole in this order, and the eluted protein solution was collected and desalted and buffered with a dialysis solution (25mM Tris pH =8.0, 10mM imidazole, 20 mM. Beta. -mercaptoethanol, 20mM cysteine, and the balance deionized water).

4) Adding TEV enzyme into the protein solution obtained in the step 3), carrying out enzymolysis for 2 hours at 25 ℃, removing a fused HIS label at the N-terminal of the protein, centrifuging and collecting supernatant. Transfer to 5mL HisFF affinity column (equilibrated with dialysate), rinse with 3 times dialysate, collect flow-through.

5) Dialyzing and desalting with dialysate (20 mM beta-mercaptoethanol, 20mM cysteine, and the balance deionized water) for 12h, and lyophilizing to obtain the target protein.

Wherein, the reconstructed recombinant keratin is marked as K31-modified (or YL 2), the unmodified keratin is marked as K31-original (or YL1, namely the keratin in CN 111202868B), and in order to compare and analyze the performance of the reconstructed recombinant keratin, the human hair extracted keratin (marked as YL 3) and the chicken and duck feather hydrolyzed keratin (marked as YL 4) are specially used as comparison. Of these, YL4 is a commercially available hydrolyzed keratin peptide (purchased from Hubei Jian peptide Biotech, inc.). YL3 is keratin extracted by the method described in the reference (Development and assessment of keratin nanoparticles for use as a systemic agent, materials Science and Engineering C63 (2016) 352-358).

EXAMPLE 2 formulation of gel dressings

The engineered recombinant keratin YL2 of example 1 was used to prepare a gel dressing.

Dissolving 1g of sodium carboxymethylcellulose and 2g of hydroxyethyl cellulose in 10g of water, and stirring to obtain a solution A;

dissolving 10g of glycerol, 10g of sorbitol and 0.8g of phenoxyethanol in water, uniformly mixing, and adding 30mg of recombinant keratin YL2 and 0.05mg of bFGF to obtain a solution B;

and adding the solution B into the solution A, stirring, and adding water to the final volume of 100mL to obtain the keratin gel dressing.

EXAMPLE 3 formulation of gel dressing

dissolving 10g of glycerol, 10g of sorbitol and 0.8g of phenoxyethanol in water, uniformly mixing, and adding 70mg of recombinant keratin YL2 and 0.05mg of bFGF to obtain a solution B;

EXAMPLE 4 formulation of gel dressing

And dissolving 30mg of recombinant keratin in water, stirring, and adding water to the final volume of 100mL to obtain the keratin gel dressing.

Comparative example 1

dissolving 10g of glycerol, 10g of sorbitol and 0.8g of phenoxyethanol in water, uniformly mixing, and adding unmodified keratin YL1 30mg and 0.05mg of bFGF to obtain a solution B;

Comparative example 2

dissolving 10g of glycerol, 10g of sorbitol and 0.8g of phenoxyethanol in water, uniformly mixing, and adding 0.05mg of bFGF to obtain a solution B;

TABLE 1 comparison of parameters for examples 2-3 and comparative examples 1-3

Examples of the experiments

Experimental example 1

Performing SDS-PAGE gel electrophoresis experiment on 2. Mu.g, 4. Mu.g and 8. Mu.g of recombinant keratin (YL 2); taking unmodified keratin (YL 1) 2 μ g, 4 μ g and 8 μ g respectively to perform SDS-PAGE gel electrophoresis experiment; the results are shown in FIG. 1. As can be seen from FIG. 1, the molecular weight of YL2 is 36kDa, and the protein purity can reach more than 95%. The purity of YL2 is higher, and the formation of dimer can be reduced, so that the expected reconstruction purpose is achieved.

Experimental example 2 cell proliferation experiment

The detection is carried out by a CCK-8 method. CCK-8 is a rapid and efficient kit for testing cell proliferation. This kit contains WST-8, which is reduced by catalase in cells to a yellow formazan product with high water solubility under the action of electron carrier 1-Methoxy PMS. The amount of formazan product produced was directly proportional to the number of living cells. Therefore, this property can be used for directly performing cell proliferation analysis. The faster the cell proliferates, the darker the color. The shade of color and the number of cells were linear for the same cells.

The specific method comprises the following steps: when fibroblasts L929 grew to 95%, cell digestion was performed, cells were centrifuged and collected, and cell suspension was prepared with DMEM medium containing serum and cell counting was performed. In 96-well plates, according to 5 x 10 ³ cell density inoculation of cells/well, volume of cell culture medium is 100. Mu.L/well. Respectively setting two groups of unmodified YL1 and modified YL2, wherein the concentration gradients of the two groups are respectively as follows: 0.005, 0.01, 0.05, 0.1, 0.14, 0.16, 0.2mg/mL. And treating the L929 cells according to the set group and concentration after the cultured cells adhere to the wall. Number ofSetting the collection time to be 0h, 24h and 48h, changing the liquid after the culture time is reached, adding 10 mu L of CCK-8 reagent and DMEM culture medium into each hole, and continuously incubating for 2 hours. And detecting the absorbance of each hole under 450nm, counting and analyzing the data, and drawing a cell growth curve.

As shown in fig. 2, the modified recombinant keratin YL2 has better cell proliferation promoting activity than the unmodified keratin YL1 under the same concentration condition.

Experimental example 3 cell migration experiment

In this experimental example 3, a cell scarification detection method is used, which is a method for simply and conveniently determining cell migration movement and repair capacity, and is similar to an in vitro wound healing model, and a microspotte head or other hard objects are used to scribe a line in the central region of cell growth on monolayer adherent cells cultured on an in vitro culture dish or a flat plate, cells in the central region are removed, then the cells are continuously cultured for a set time (for example, 24 hours) in an experiment, a cell culture plate is taken out, whether peripheral cells migrate (repair) to a central scarification region or not is observed, and thus the growth migration capacity of the cells is judged.

The specific method comprises the following steps: the fibroblast cells L929 were digested when they were 95% grown, centrifuged at 1000rpm for 5min and collected, and the cell suspension was prepared with serum-containing DMEM complete medium and cell counting was performed. According to 3 x 10 ⁵ cell/well inoculated into 6-well plates at a volume of 1.5mL,37 ℃ and 5% CO ₂ After the cells are cultured in the incubator and attached to the wall, a blank area is created by vertically aligning a 200-L gun head with the bottom of the 6-well plate and drawing a cross. After the floating cells were washed by adding PBS buffer, DMEM basal medium was added. The following 4 groups were set: the modified recombinant keratin YL2, the unmodified keratin YL1 and human hair are extracted to obtain keratin YL3 and hydrolyzed keratin YL4 which is commercially available. Each group is respectively provided with a concentration gradient as follows: 0.05, 0.1, 0.2, 0.4, 0.6, 0.8mg/mL. And treating the cells in different holes according to different concentration gradients of different groups, and taking pictures in the same area by using microscope equipment when the treatment time is 0h, 24h, 48h and 72 h. The area of cell migration was analyzed by ImageJ software. The cell healing rate was calculated according to the formula to express the migration rate of the cells.

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

As the results are shown in fig. 3 to 6, it can be seen that the efficacy of the K31 keratin after the modification, i.e., YL2, in promoting cell migration is significant, superior to the keratin YL1, YL3 and YL4 before the modification. In addition, it can be seen that the effect is particularly remarkable when the concentration of the recombinant keratin is 0.1 mg/mL.

EXAMPLE 4 wound repair

Selecting the established rat diabetes model, and establishing a wound model for the diabetic rat: after rats are anesthetized, unhaired and sterilized by 75% ethanol, a wound surface with 1.5X 1.5cm of full-layer skin defect is cut by surgical scissors to reach the fascia layer without cutting muscle layer and blood vessel. The settings are as follows: blank control (without any dressing), dressing from example 4 was applied correspondingly, dressing from example 2 was applied correspondingly, and dressings from comparative example 1 and comparative example 2 were applied correspondingly. The wound is bandaged by medical bandage and gauze, so as to avoid wound infection. Photographs of the wounds were taken at different time points (

day

0, 3, 6, 9, 11, 14, 18) after the operation, and the healing rate of each wound was analyzed and calculated by observing the degree of healing of the wound surface and using ImageJ software. The calculation formula of the healing rate is as follows:

wherein A is ₀ The initial area of the wound surface, A _t Wound area on day "t" post-trauma.

The wound repair results are shown in fig. 7-10, and the recombinant keratin gel dressing has a remarkable healing promotion effect in the wound repair process.

From the perspective of wound healing, the wound healing rat model maps of examples 2 and 4 were superior, with short healing time and high healing rate, compared to comparative examples 1-2 and the blank control group. In addition, the wound healing effect of example 2 is superior to that of example 4.

Compared with the comparative example 1, the comparative example 2 is not added with keratin, the wound repair process is much slower than that of the comparative example 1, the wound is also more serious than that of the comparative example 1, and the healing rate is lower than that of the comparative example 1, which shows that the keratin plays a certain positive role in wound healing.

SEQUENCE LISTING

<110> Haimeisi (Chongqing) medical Biotechnology Ltd

<120> keratin, keratin gel dressing for chronic wound repair, preparation method and application thereof

<130> TPF02217

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<170> PatentIn version 3.5

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Arg Glu Val Glu Gln Trp Phe Thr Thr Gln Thr Glu Glu Leu Asn Lys

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Gln Val Val Ser Ser Ser Glu Gln Leu Gln Ser Tyr Gln Ala Glu Ile

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gaaccgctgc tgtgcccgag ctaccagagc tattttaaaa ccattgaaga actgcagcag 180

aaaattctgt gcaccaaaag cgaaaacgcg cgcctggttg tacagattga taacgccaaa 240

ctggcggccg atgatttccg caccaaatat cagaccgaac tgagcctgcg ccagctggtg 300

gaaagcgata ttaacggtct gcgccgtatc ctggatgaac tgaccctgtg caaatccgat 360

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caggaagtga acaccctgcg ctgccagctg ggcgatcgtc tgaatgtgga ggtggatgcg 480

gccccgacgg tggatctgaa ccgcgtgctg aacgaaaccc gtagccaata tgaagcgctg 540

gtggaaacca accgtcgtga agtggaacag tggtttacga ctcagaccga agaactgaat 600

aaacaggtgg tgagtagctc agaacagctg cagtcatatc aggccgaaat cattgaactg 660

cgccgcaccg tgaacgcgct ggaaattgaa ctgcaggccc agcacaatct gcgtgatagc 720

ctggaaaata ccctgaccga aagcgaagcg cgctatagca gccagctgag ccaggtacag 780

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Claims

1. A keratin, characterized in that the amino acid sequence of the keratin is shown as SEQ ID No. 2.

2. A nucleic acid sequence encoding the keratin of claim 1.

3. The nucleic acid sequence of claim 2, wherein the nucleic acid sequence is set forth in SEQ ID No. 3.

4. A keratin gel dressing comprising the keratin of claim 1;

5. The keratin gel dressing of claim 4, further comprising a basic fibroblast growth factor (bFGF) and a gel matrix;

preferably, the gel matrix is selected from one or two of sodium carboxymethylcellulose and hydroxyethylcellulose; further preferably, the gel matrix is sodium carboxymethyl cellulose and hydroxyethyl cellulose.

6. The keratin gel dressing of claim 5,

the mass ratio of the basic fibroblast growth factor in the keratin gel dressing is 0.01-1 mu g/g;

preferably, the weight percentage of the sodium carboxymethyl cellulose is 0.25-1.25wt%, and the weight percentage of the hydroxyethyl cellulose is 0.5-2.5wt%, based on the total weight of the keratin gel dressing.

7. The keratin gel dressing of claim 4,

the keratin gel dressing further comprises a humectant, a preservative and a thickener;

preferably, the first and second liquid crystal display panels are,

the humectant is selected from one or more of glycerol, propylene glycol and silane;

the preservative is phenoxyethanol;

the thickening agent is sorbitol;

it is further preferred that the first and second liquid crystal display panels,

8. A method for preparing a keratin gel dressing,

dissolving the gel matrix in water, and stirring to obtain a solution A;

dissolving a humectant, a thickening agent and a preservative in water, uniformly mixing, and adding keratin and basic fibroblast growth factor to obtain a solution B;

adding the solution B into the solution A, stirring, adding water to obtain keratin gel dressing,

the amino acid sequence of the keratin is shown as SEQ ID No. 2.

9. Use of a keratin protein according to claim 1, or a keratin gel dressing according to any one of claims 4 to 7, or a keratin gel dressing prepared by a method according to claim 8, in the manufacture of a medicament for promoting wound healing;

10. Use of the keratin of claim 1, or the keratin gel dressing of any one of claims 4 to 7, or the keratin gel dressing produced by the method of claim 8 in the production of a vascular endothelial cell proliferation promoting agent.

11. A medicament for promoting wound healing or vascular endothelial cell proliferation, comprising the keratin of claim 1 or the keratin expressed by the nucleic acid sequence of claim 2 or 3 or the keratin gel dressing of any one of claims 4 to 7.