Recombinant collagen-like protein containing mussel mucoprotein sequence and application of recombinant collagen-like protein in skin mucous membrane repair and tissue tightening

Abstract

The invention provides a protein. The protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-9. Furthermore, the amino acid sequence of the protein is as shown in SEQ ID NO: 8 or 9. The invention provides a recombinant collagen-like protein containing a mussel mucin sequence. The mussel mucoprotein is high in viscosity, and on the basis of physics, collagen is added to repair damage, so that the function of the fusion protein is improved. The invention also provides application of the protein in skin mucous membrane repair and tissue tightening.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a recombinant collagen-like protein and its application in skin and mucous membrane repair and tissue tightening. Background Art

[0002] Skin and mucous membranes are crucial barrier systems for the human body, not only defending against the invasion of harmful external factors but also playing a vital role in maintaining homeostasis, regulating temperature, and sensing stimuli. However, under the influence of various factors such as trauma, aging, childbirth, and disease, skin and mucous membranes are prone to damage or structural and functional degradation, leading to a series of clinical or sub-health problems.

[0003] Skin injury repair remains a core challenge in fields such as surgery, dermatology, burns, and diabetic foot. Traditional repair strategies rely primarily on antibiotic ointments, physiological healing, and artificial dressings. However, these approaches often fail to accelerate tissue reconstruction, are prone to scarring, and have limited repair quality. In recent years, bioactive proteins have garnered widespread attention due to their ability to stimulate fibroblast proliferation, induce angiogenesis, and promote matrix remodeling.

[0004] Furthermore, tissue aging phenomena such as skin laxity, facial sagging, and vaginal laxity are common concerns for the modern population, especially for middle-aged and elderly people and postpartum women, seriously impacting their quality of life. Skin laxity is primarily related to fibroblast dysfunction, collagen loss, and elastic fiber fragmentation. While existing medical aesthetics treatments such as radiofrequency, laser, and hyaluronic acid fillers can temporarily improve appearance, they lack the ability to fundamentally activate skin structural reconstruction, resulting in limited and short-lived results.

[0005] Hemorrhoids are another disease closely related to loose mucosal and connective tissue structures, primarily caused by degeneration of the anal cushion support structure, varicose veins, and decreased elasticity of the submucosal tissue. Clinical treatment typically involves surgery or hormonal ointments, but these can lead to slow recovery and a high recurrence rate. Hormonal preparations can also cause local irritation and long-term dependency.

[0006] Recent studies have demonstrated that certain proteins or fusion proteins derived from specific sources have demonstrated promising effects on promoting fibroblast proliferation, epithelial tissue repair, and collagen remodeling, accelerating healing, increasing tissue density, and reducing scarring in animal trauma models. The application of these proteins in skin and mucosal repair and tissue reconstruction is expected to become a novel bioactive strategy for addressing these diverse issues, with promising market potential and clinical prospects.

[0007] However, there is still a lack of proteins with high activity in skin damage repair and other aspects. Therefore, it is urgent to develop a protein with high biological activity to meet the dual needs of clinical and daily beauty. Summary of the Invention

[0008] In a first aspect of the present invention, a protein is provided, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-9.

[0009] In some embodiments of the present invention, the amino acid sequence of the protein is shown in any one of SEQ ID NOs: 1-9.

[0010] In another aspect of the present invention, a protein is provided, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5, 6, 8 and 9.

[0011] In some embodiments of the present invention, the protein comprises the amino acid sequence shown in SEQ ID NO: 8 or 9.

[0012] In some embodiments of the present invention, the protein comprises the amino acid sequence shown in SEQ ID NO:8.

[0013] In some embodiments of the present invention, the protein comprises the amino acid sequence shown in SEQ ID NO:9.

[0014] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO: 5, 6, 8 or 9.

[0015] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO: 8 or 9.

[0016] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO:8.

[0017] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO:9.

[0018] In some embodiments of the present invention, the protein is any protein described in Example 2 of the present invention.

[0019] In some embodiments of the present invention, the protein is the modified fusion protein 2 as described in Example 2 of the present invention.

[0020] In some embodiments of the present invention, the preparation method of the modified fusion protein 2 is as follows: the raw materials are mixed and dissolved in ultrapure water to obtain 30 mL of solution, so that the solution contains 1 mg / mL of fusion protein 2, 15 mmol / L ascorbic acid, 20 μmol / L copper sulfate, 20 mmol / L phosphate buffer at pH 7.6 and 1500 U of tyrosinase; stirring at room temperature for 4 hours, modifying the tyrosine residue of fusion protein 2 to DOPA, adding glacial acetic acid at the modification end point to adjust the pH of the feed solution to 3.3; the reaction solution is purified by ion exchange chromatography, the eluate is desalted and concentrated, the retentate is collected, and finally the retentate is purified again by ion exchange chromatography, and the eluate is freeze-dried to obtain a pure lyophilized powder of the modified fusion protein 2.

[0021] In another aspect of the present invention, a nucleic acid is provided, comprising a nucleotide sequence encoding the protein of the present invention.

[0022] In some embodiments of the present invention, the nucleic acid comprises a nucleotide sequence encoding a protein whose amino acid sequence is shown in any one of SEQ ID NOs: 5, 6, 8 and 9.

[0023] In some embodiments of the present invention, the nucleic acid comprises a nucleotide sequence encoding a protein whose amino acid sequence is shown in SEQ ID NO:5.

[0024] In some embodiments of the present invention, the nucleic acid comprises a nucleotide sequence encoding a protein whose amino acid sequence is shown in SEQ ID NO:6.

[0025] In some embodiments of the present invention, the nucleic acid comprises a nucleotide sequence encoding a protein whose amino acid sequence is shown in SEQ ID NO:8.

[0026] In some embodiments of the present invention, the nucleic acid comprises a nucleotide sequence encoding a protein whose amino acid sequence is shown in SEQ ID NO:9.

[0027] In some embodiments of the present invention, the nucleic acid is an RNA sequence or a DNA sequence.

[0028] In some embodiments of the invention, the nucleic acid is a DNA nucleic acid. In some embodiments of the invention, the nucleic acid is an RNA nucleic acid.

[0029] In some embodiments of the present invention, the nucleic acid is a DNA nucleic acid, including but not limited to plasmid DNA, linear DNA, double-stranded DNA, or synthetic DNA fragments. Preferably, it is prepared by molecular cloning, chemical synthesis, or PCR amplification, and may incorporate regulatory elements such as promoters, enhancers, multiple cloning sites, selectable marker genes, and transcription terminators to enhance expression levels in host cells.

[0030] In some embodiments of the present invention, the nucleic acid is an RNA nucleic acid, including but not limited to mRNA. Preferably, the mRNA comprises a 5' cap structure (Cap 0 / Cap 1), an optimized 5'-UTR, an open reading frame (ORF), an optimized 3'-UTR, and a poly(A) tail to enhance stability and translation efficiency in eukaryotic cells.

[0031] In certain embodiments, the nucleotide sequence is codon-optimized to enhance expression in specific host cells. Optimization is based on the codon usage bias of highly expressed genes in the target system, reducing the probability of mRNA secondary structure formation and promoting translation initiation and elongation efficiency. Further optimization may include avoiding rare codons, reducing repetitive sequences, and eliminating potential splice sites, RNA editing sites, stop codons, RNAi targets, and restriction enzyme sites.

[0032] The nucleic acid can incorporate regulatory elements suitable for various expression systems, such as the Escherichia coli T7 promoter-lac operon, the Pichia pastoris AOX1 promoter, or the mammalian CMV or EF-1α promoter. It can be delivered via transfection, transformation, transduction, or microinjection to any of the following cells: prokaryotic cells (e.g., Escherichia coli BL21(DE3), Rosetta(DE3)); yeast cells (Pichia pastoris, Saccharomyces cerevisiae); mammalian cells (CHO, HEK293, HepG2); plant cells (Nicotiana tabacum BY-2); insect cells (Sf9 / Sf21 combined with baculovirus system); fungal or microalgal cells. Preferably, vectors such as pET, pcDNA, or pPIC are used in combination with gene editing (e.g., nuclease gene knockout) to enhance expression efficiency.

[0033] In another aspect of the present invention, a vector is provided, wherein the vector comprises the nucleic acid of the present invention.

[0034] In some embodiments of the present invention, the vector comprises a nucleotide sequence encoding the protein of the present invention.

[0035] In another aspect of the present invention, a vector is provided, comprising the nucleic acid of the present invention, and can be used to express a target nucleic acid sequence in a host cell, thereby producing a desired protein or functional RNA product.

[0036] In some embodiments of the present invention, the vector comprises a nucleotide sequence encoding the protein of the present invention, and is capable of driving the transcription and / or translation of the sequence in a suitable expression system.

[0037] In some embodiments of the present invention, the vector is a plasmid vector, preferably a circular double-stranded DNA structure, which has the characteristics of high copy number, strong stability, and simple operation, and is suitable for prokaryotic or eukaryotic expression systems.

[0038] In some embodiments of the present invention, the plasmid vector includes but is not limited to the following vector types:

[0039] The pGEX vector series, such as pGEX-4T-1, can be used for fusion expression of GST-tagged proteins to facilitate affinity purification; the pET vector series, such as pET-30a(+), pET-21a(+), pET-28a(+), etc., are generally used for efficient expression of target proteins in Escherichia coli; the pUC vector series, such as pUC19 vector, is often suitable for cloning and initial expression construction; the pYES2 series vectors are suitable for yeast expression; the pBAD vector series, such as pBAD24, is used for controllable expression induced by arabinose; mammalian expression vectors such as pcDNA3.1 and pCMV series.

[0040] In some embodiments of the present invention, the vector is a viral vector, which can be used to efficiently transduce various eukaryotic cells, and is particularly suitable for cell types that are difficult to transfect, or for in vivo delivery.

[0041] In some embodiments of the present invention, the viral vector includes but is not limited to:

[0042] Adeno-associated virus (AAV) vectors have low immunogenicity and can express in vivo for a long time, making them suitable for gene therapy applications. Common serotypes include AAV1, AAV3, AAV4, AAV5, AAV2, AAV6, AAV8, and AAV9. Adenovirus (AV) vectors have high titers and high infection efficiency and are commonly used for in vitro experiments and some short-term in vivo expression. Lentivirus vectors can integrate into the host genome, achieving long-term stable expression and are widely used in mammalian cell line or stem cell research.

[0043] In some embodiments of the present invention, the vector may also contain a His tag, a fluorescent tag (such as EGFP, mCherry), or a reporter gene (such as Luciferase, β-Galactosidase) for in vivo imaging, purification, expression tracking, or functional verification; the tag may be located at the N-terminus or C-terminus, or connected to the target protein via a flexible linker peptide, such as a Gly-Ser repeat peptide (such as GGGGS). Optionally, a protease cleavage site (such as TEV, Thrombin, FactorXa site) is set between the tag and the target protein to remove the tag after purification.

[0044] In another aspect of the present invention, a cell is provided, wherein the cell comprises the protein, nucleic acid or vector of the present invention.

[0045] The cells can be used to produce, express, carry or deliver the nucleic acid or protein of the present invention; this is applicable to multiple application fields such as basic research, formulation screening, vaccine development, gene therapy, and recombinant protein production.

[0046] In some embodiments of the present invention, the cell is a recombinant cell, which has been introduced with an exogenous expression construct (such as a plasmid vector, a viral vector, etc.) containing a target nucleic acid sequence by transfection, transduction or transformation, and can express the protein or functional RNA product of the present invention under in vivo or in vitro conditions.

[0047] In some embodiments of the present invention, the cells are mammalian cells, including but not limited to: HEK293 cells, human embryonic kidney cells, suitable for transient transfection and virus packaging; CHO cells, Chinese hamster ovary cells, suitable for large-scale production of glycosylated recombinant proteins; BHK cells, Vero cells, C2C12 cells, NS0 cells, HepG2 cells, HuH-7 cells, HUDEP cells, etc.

[0048] In some embodiments of the present invention, the cells are subjected to gene knockout (KO), gene knockin (KI) or humanization transformation to improve expression stability and yield or meet formulation development requirements.

[0049] In some embodiments of the present invention, the cells are prokaryotic cells, such as Escherichia coli (E. coli) expression strains, including but not limited to Rosetta (DE3), DH5α, TOP10, BL21 (DE3), JM109, etc., which are suitable for the expression of non-glycosylated proteins or short peptides.

[0050] In some embodiments of the present invention, the cell is a yeast cell, such as Pichia pastoris or Saccharomyces cerevisiae, and the yeast cell can be used for high-density fermentation expression or secretory protein production.

[0051] In some embodiments of the present invention, the cells are insect cells, such as Sf9, Sf21 or High Five cells, which are suitable for efficient expression of recombinant proteins in a baculovirus expression system.

[0052] In some embodiments of the present invention, the cells are plant cells, such as tobacco BY-2 suspension cells, or plant cells in a plant leaf transient expression system mediated by Agrobacterium.

[0053] In some embodiments of the present invention, the cells are viral packaging cell lines used to produce viral particles containing the nucleic acid of the present invention, for example, HEK293-AAV cells for AAV packaging, HEK293T cells for lentivirus packaging, 293A cells for adenovirus packaging, etc.

[0054] In some embodiments of the present invention, the protein expressed in the cell may contain a tag sequence to facilitate subsequent purification, detection or functional studies. Alternatively, the cell may stably express the target protein or transiently express the target protein.

[0055] In some embodiments of the present invention, the cells also have one or more of the following characteristics: the expression product has correct post-translational modification (such as glycosylation, phosphorylation); the expression system is safe and controllable, suitable for clinical research or production; high expression level; the expression product can be secreted into the culture medium; and large-scale amplification culture can be performed.

[0056] In another aspect of the present invention, a drug is provided, comprising the protein, nucleic acid or vector of the present invention.

[0057] In some embodiments of the present invention, the medicament comprises the protein of the present invention.

[0058] In some embodiments of the present invention, the drug comprises a protein having an amino acid sequence as shown in SEQ ID NO:5.

[0059] In some embodiments of the present invention, the drug comprises a protein having an amino acid sequence as shown in SEQ ID NO:6.

[0060] In some embodiments of the present invention, the drug comprises a protein having an amino acid sequence as shown in SEQ ID NO:8.

[0061] In some embodiments of the present invention, the drug comprises a protein having an amino acid sequence as shown in SEQ ID NO:9.

[0062] In some embodiments of the present invention, the medicament comprises a pharmaceutically acceptable excipient.

[0063] In some embodiments of the present invention, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients to enhance the stability, delivery efficiency, bioavailability, or formulation suitability of the active ingredient. The excipients should comply with international pharmacopoeia standards (e.g., USP / EP / ChP) and have no significant toxic or immunogenic properties.

[0064] In some embodiments of the present invention, the excipients specifically include but are not limited to the following categories:

[0065] Carriers and excipients: diluents / fillers such as carbohydrates: lactose (anhydrous / monohydrate), sucrose, mannitol, sorbitol, microcrystalline cellulose (such as PH series), pregelatinized starch; inorganic salts such as calcium phosphate (dihydrate / anhydrous), calcium carbonate, calcium sulfate; proteins such as gelatin, hydrolyzed collagen; adhesives such as synthetic polymers polyvinylpyrrolidone (PVP K30 / K90), polyethylene glycol (PEG 4000-6000), natural polymers hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), methylcellulose, gum arabic, carbohydrate derivatives trehalose, maltodextrin;

[0066] Functional excipients: disintegrants such as cross-linked carboxymethyl cellulose sodium (CCNa), cross-linked polyvinylpyrrolidone (PVPP), sodium starch glycolate (SSG), low-substituted hydroxypropyl cellulose (L-HPC); lubricants / glidants such as metal stearates (magnesium stearate / calcium / zinc stearate), glyceryl behenate ( 888), talc, colloidal silicon dioxide Surfactants such as ionic sodium dodecyl sulfate (SDS), bile salts, and non-ionic surfactants such as polysorbate ( 20 / 80), Poloxamer (Poloxamer 188 / 407), Span ( 80);

[0067] Stabilizers and protective agents: antioxidants such as water-soluble ascorbic acid, sodium thiosulfate, and methionine; oil-soluble tocopherol (vitamin E), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ); chelating agents such as disodium / calcium EDTA, citric acid, and tartaric acid; freeze-drying protective agents (applicable to biological products): sugars such as sucrose, trehalose, and mannitol; amino acids such as glycine and arginine; and polymers such as dextran and povidone.

[0068] Delivery system excipients: sustained-release materials such as hydrophilic gel carbomer Chitosan, enteric coatings such as hydroxypropyl methylcellulose phthalate (HPMCP) and hydroxypropyl methylcellulose acetate succinate (HPMCAS); transdermal enhancers such as azone, oleic acid, and terpenes (menthol / limonene); nanocarrier materials such as liposomes, phosphatidylcholine, and cholesterol; and polymer nanoparticles such as PLGA and chitosan.

[0069] Special dosage form excipients: Excipients for injections such as isotonicity regulators: sodium chloride, glycerol, pH buffers: phosphate buffered saline (PBS), citrate buffer, Tris-HCl, antibacterial agents: benzyl alcohol, phenol, m-cresol (must comply with injection limits); excipients for biomacromolecule preparations such as anti-aggregants: polysorbate 80, trehalose, enzyme inhibitors such as aprotinin and leupeptin.

[0070] In another aspect of the present invention, a preparation is provided, comprising the protein, nucleic acid or vector of the present invention.

[0071] In some embodiments of the present invention, the formulation comprises a protein of the present invention.

[0072] In some embodiments of the present invention, the preparation comprises a protein having an amino acid sequence as shown in SEQ ID NO:5.

[0073] In some embodiments of the present invention, the formulation comprises a protein having an amino acid sequence as shown in SEQ ID NO:6.

[0074] In some embodiments of the present invention, the formulation comprises a protein having an amino acid sequence as shown in SEQ ID NO:8.

[0075] In some embodiments of the present invention, the formulation comprises a protein having an amino acid sequence as shown in SEQ ID NO:9.

[0076] In some embodiments of the present invention, the formulation comprises a pharmaceutically acceptable excipient.

[0077] In some embodiments of the present invention, the excipients specifically include but are not limited to the following categories:

[0078] Carriers and excipients: diluents / fillers such as carbohydrates: lactose (anhydrous / monohydrate), sucrose, mannitol, sorbitol, microcrystalline cellulose (such as PH series), pregelatinized starch; inorganic salts such as calcium phosphate (dihydrate / anhydrous), calcium carbonate, calcium sulfate; proteins such as gelatin, hydrolyzed collagen; adhesives such as synthetic polymers polyvinylpyrrolidone (PVP K30 / K90), polyethylene glycol (PEG 4000-6000), natural polymers hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), methylcellulose, gum arabic, carbohydrate derivatives trehalose, maltodextrin;

[0079] Functional excipients: disintegrants such as cross-linked carboxymethyl cellulose sodium (CCNa), cross-linked polyvinylpyrrolidone (PVPP), sodium starch glycolate (SSG), low-substituted hydroxypropyl cellulose (L-HPC); lubricants / glidants such as metal stearates (magnesium stearate / calcium / zinc stearate), glyceryl behenate ( 888), talc, colloidal silicon dioxide Surfactants such as ionic sodium dodecyl sulfate (SDS), bile salts, and non-ionic surfactants such as polysorbate ( 20 / 80), Poloxamer (Poloxamer 188 / 407), Span ( 80);

[0080] Stabilizers and protective agents: antioxidants such as water-soluble ascorbic acid, sodium thiosulfate, and methionine; oil-soluble tocopherol (vitamin E), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ); chelating agents such as disodium / calcium EDTA, citric acid, and tartaric acid; freeze-drying protective agents (applicable to biological products): sugars such as sucrose, trehalose, and mannitol; amino acids such as glycine and arginine; and polymers such as dextran and povidone.

[0081] Delivery system excipients: sustained-release materials such as hydrophilic gel carbomer Chitosan, enteric coatings such as hydroxypropyl methylcellulose phthalate (HPMCP) and hydroxypropyl methylcellulose acetate succinate (HPMCAS); transdermal enhancers such as azone, oleic acid, and terpenes (menthol / limonene); nanocarrier materials such as liposomes, phosphatidylcholine, and cholesterol; and polymer nanoparticles such as PLGA and chitosan.

[0082] Special dosage form excipients: Excipients for injections such as isotonicity regulators: sodium chloride, glycerol, pH buffers: phosphate buffered saline (PBS), citrate buffer, Tris-HCl, antibacterial agents: benzyl alcohol, phenol, m-cresol (must comply with injection limits); excipients for biomacromolecule preparations such as anti-aggregants: polysorbate 80, trehalose, enzyme inhibitors such as aprotinin and leupeptin.

[0083] In another aspect of the present invention, there is provided the use of the protein, nucleic acid or vector of the present invention in the preparation of a drug for promoting skin damage repair, promoting skin firming, treating hemorrhoids, relieving vaginal relaxation, treating skin laxity or promoting fibroblast proliferation.

[0084] In some embodiments of the present invention, there is provided use of the protein of the present invention in the preparation of a medicament for promoting skin damage repair, promoting skin firming, treating hemorrhoids, alleviating vaginal laxity, treating skin laxity, or promoting fibroblast proliferation.

[0085] In some embodiments of the present invention, there is provided use of the protein of the present invention in the preparation of a medicament for promoting the repair of skin damage.

[0086] In some embodiments of the present invention, there is provided use of the protein of the present invention in the preparation of a medicament for promoting the repair of skin and mucosal injuries.

[0087] In some embodiments of the present invention, there is provided use of the protein of the present invention in the preparation of a medicament for promoting fibroblast proliferation.

[0088] In some embodiments of the present invention, there is provided use of the protein of the present invention in the preparation of a medicament for promoting skin firming.

[0089] In some embodiments of the present invention, there is provided use of the protein of the present invention in the preparation of a medicament for treating hemorrhoids.

[0090] In some embodiments of the present invention, there is provided use of the protein of the present invention in the preparation of a medicament for alleviating vaginal relaxation.

[0091] In some embodiments of the present invention, there is provided use of the protein of the present invention in the preparation of a medicament for treating cutis laxity.

[0092] The protein of the present invention has been experimentally demonstrated to be highly active in promoting fibroblast proliferation and wound healing in animals. Furthermore, those skilled in the art are aware that fibroblasts play a central role in tissue repair: they are the primary structural cells of the dermis, responsible for synthesizing matrix components such as collagen, elastin, and hyaluronic acid. During wound repair, fibroblasts participate in wound contraction, granulation tissue formation, and skin regeneration.

[0093] Therefore, the protein of the present invention can be used to promote the repair of skin injuries, such as surgical incisions, abrasions, burns, diabetic foot, bedsores, etc.; the protein of the present invention can be used to promote skin firming, and its mechanism of action is to stimulate the proliferation of fibroblasts, thereby increasing the synthesis of collagen, elastin and hyaluronic acid, and improving skin elasticity and tension; the protein of the present invention can be used to treat hemorrhoids, and the principle is that hemorrhoids are essentially venous dilation and connective tissue relaxation in the anal cushion tissue. Protein-induced tissue repair and elasticity reconstruction can help improve symptoms; the protein of the present invention can be used to relieve vaginal relaxation. Vaginal mucosal relaxation caused by childbirth or aging can lead to sexual discomfort and urinary incontinence. The protein of the present invention can restore firmness by promoting collagen regeneration and vascular reconstruction in the vaginal submucosal tissue; the protein of the present invention has the activity of promoting fibroblast proliferation and promoting skin wound healing, and therefore can be used to treat skin relaxation.

[0094] Without being bound by any particular theory, the inventors discovered that even without DOPA modification, fusion protein 2 exhibited similar or comparable fibroblast proliferation and tissue adhesion activities as the modified protein. This phenomenon may be due to the introduction of multiple tyrosine (Tyr, Y) residues into the Mfp-2 variant and the partial Mfp-5 peptide variant contained in fusion protein 2. The resulting phenolic hydroxyl structure can, to a certain extent, mimic the dihydroxy aromatic ring structure of DOPA, thereby partially replacing its ability to interact with substrate surfaces or tissue proteins.

[0095] Furthermore, the aforementioned Mfp-2 variant and Mfp-5 peptides possess inherently strong adhesion capabilities, potentially creating a synergistic effect with collagen-derived peptides, thereby enhancing the overall protein's adhesion and bioactivity on moist tissue surfaces. This synergistic effect may help the protein maintain robust activity without the need for enzymatic modification, broadening its applicability and ease of preparation. DETAILED DESCRIPTION

[0096] In the present invention, the terms mussel mucin and mussel mucin are used interchangeably.

[0097] The present disclosure is further illustrated by the following examples, but is not intended to limit the present disclosure to these examples. Experimental methods in the following examples, where specific conditions are not specified, were performed according to conventional methods and conditions, or as selected from commercial product specifications. Unless otherwise noted, reagents and raw materials used in this disclosure are commercially available.

[0098] Example 1: Protein Design

[0099] The inventors designed and tested human type 1 collagen, mussel mucin, and their fusion proteins, as shown in Table 1.

[0100] Table 1. Designed proteins

[0101]

[0102]

[0103] Among them, partial peptides of human type 1 collagen were recombinantly fused with variants of mussel mucin Mfp-2 and mussel mucin Mfp-5 to express as recombinant collagen-like protein (fusion protein 2), named as adhesive protein, English name CMFP1.x, registered trademark Baoyintai, and R&D code FLM3.6.x.

[0104] Example 2: Preparation of protein

[0105] A DNA nucleic acid fragment containing the coding nucleotide sequence of the fusion protein 2 described in Example 1 (sequence as shown in SEQ ID NO: 8) was prepared. The nucleotide sequence had been codon-optimized for Escherichia coli preference expression using ExpOptimizer, using default parameters. The nucleic acid fragment was digested with XbaI and BlpI enzymes. The pET28a(+) vector was then taken and digested with XbaI and BlpI enzymes, respectively. The two enzyme digestion reaction systems were connected using T4 DNA ligase. The ligation product was transformed into stbl3 competent cells, cultured for 15 hours, and positive single clones were selected for sequencing identification. After the positive clones with correct sequencing were cultured normally, the expression vector plasmid (SEQ ID NO: 10) of the fusion protein 2 was extracted.

[0106] The expression vector plasmid for fusion protein 2 was transformed into the expression strain E. coli BL21(DE3). Cultures were grown in LB broth containing kanamycin at 37°C for 9 hours. IPTG was added to 0.25 mM and induced at 16°C for 20 hours. The cells were harvested by centrifugation, lysate was added, and the supernatant was obtained by centrifugation. After filtration through a 0.45 μm filter, the supernatant was loaded onto a Ni-NTA column. Contaminants were first washed away with elution buffer, followed by elution of the target protein with buffer. Desalting was then performed, followed by reverse-phase HPLC purification using a C18 column (flow rate 1 mL / min, gradient elution using 0-70% acetonitrile, 0.1% TFA). The target peak was collected and freeze-dried to obtain a fusion protein 2 powder with a purity of 96.5%. SDS-PAGE analysis of this sample revealed a single band, and LC-MS confirmed that the molecular weight was consistent with the theoretical value.

[0107] The same method was used to prepare the other proteins in Table 1: only the protein coding sequences were different, and the rest were the same. The expression vector plasmids of each protein were prepared, and then the proteins were prepared and purified with a purity of 95.5% to 96.7%.

[0108] Preparation of modified fusion protein 2: All raw materials were mixed and dissolved in ultrapure water to yield a 30 mL solution containing 1 mg / mL fusion protein 2, 15 mmol / L ascorbic acid, 20 μmol / L copper sulfate, 20 mmol / L phosphate buffer (pH 7.6), and 1500 U tyrosinase. The solution was stirred at room temperature for 4 hours. The tyrosine residues of fusion protein 2 were modified with DOPA. At the end of the modification, glacial acetic acid was added to adjust the pH of the solution to 3.3. The reaction solution was purified by ion exchange chromatography, the eluate was desalted and concentrated, and the retentate was collected. Finally, the retentate was purified again by ion exchange chromatography. The eluate was freeze-dried to obtain a pure lyophilized powder of modified fusion protein 2 with a purity of 94.6%.

[0109] Example 3: Cell proliferation activity test

[0110] 1×10^5 L929 cells were plated in a 24-well plate. Each protein prepared as described in Example 2 was dissolved in sterile PBS and sterilized through a 0.22μm filter before addition to the plate to a total protein concentration of 200 μg / ml. (No protein was added to the negative control group.) After 60 hours of incubation in DMEM medium supplemented with 10% FBS at 37°C and 5% CO₂, the cells were assayed using the MTT assay, with OD570 absorbance as an indicator of cell proliferation activity. The assay was repeated five times, and the average results are shown in Table 2. The OD570 values ​​of the Fusion Protein 2 and Modified Fusion Protein 2 experimental groups were significantly higher than those of the other groups, demonstrating statistical significance (P<0.001). The OD570 value of the fusion protein 3 experimental group was significantly higher than that of the Mfp-2 experimental group, the Mfp-5 partial peptide experimental group and the human type 1 collagen partial peptide (B), with statistically significant differences (P<0.05).

[0111] Fusion protein 2 significantly promotes L929 cell proliferation. The partial peptide fragment (A) of type 1 collagen, the Mfp-2 variant, and the partial peptide fragment variant of Mfp-5 in fusion protein 2 are all crucial for its activity. When any component of the fusion protein is replaced with another peptide, its proliferative activity is significantly reduced. Furthermore, compared with a simple mixture of the corresponding components, fusion protein 2 exhibits higher activity, suggesting that its functional activity may be enhanced through a specific three-dimensional conformation or interdomain synergy.

[0112] Table 2. Cell proliferation activity test results

[0113]

[0114]

[0115] Example 4: Animal Skin Wound Healing Test

[0116] Healthy, male Sprague-Dawley (SD) rats weighing 205-209 g were randomly divided into 10 groups per group. Under intravenous anesthesia, the back was depilated, and a 1 cm × 1 cm full-thickness excisional wound was created on the left back. The experimental group received 100 μL of gel applied to the wound once daily. The gel was prepared by mixing glycerol (6%), triethanolamine (1%), carbomer 940 (0.1%), any protein (0.3% by weight) or a mixture of proteins prepared as described in Example 2 (total 0.3% by weight), and the remainder of water. The gel was sterilely prepared and stored at 4°C in the dark. The blank group received no treatment and allowed to heal naturally. On day 7, the wounds were aligned with a ruler and photographed. The area was calculated using image analysis software, and the mean value for each group was used. The results are shown in Table 3. The wound areas in the fusion protein 2 and modified fusion protein 2 groups were significantly lower than those in the other groups, with statistically significant differences (P < 0.001). This shows that fusion protein 2 has a strong effect in promoting wound healing and has achieved unexpected technical results.

[0117] Table 3. Animal skin wound healing test results

[0118]

[0119]

Claims

1. A protein, characterized in that The protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5, 6, 8 and 9.

2. A protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO: 5, 6, 8 or 9.

3. A protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO: 8 or 9.

4. A protein, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO:

8.

5. A nucleic acid, characterized in that The nucleic acid comprises a nucleotide sequence encoding the protein according to claim 2.

6. A carrier, characterized in that The vector comprises the nucleic acid according to claim 5.

7. A preparation, characterized in that The cell comprises the protein of claim 2, the nucleic acid of claim 5 or the vector of claim 6.

8. Use of the protein according to claim 2, the nucleic acid according to claim 5, or the vector according to claim 6 in the preparation of a medicament for promoting skin damage repair, promoting skin firming, treating hemorrhoids, alleviating vaginal laxity, treating skin laxity, or promoting fibroblast proliferation.

9. Use of the protein according to claim 2 in the preparation of a medicament for promoting the repair of skin damage.

10. Use of the protein according to claim 2 in the preparation of a medicament for promoting fibroblast proliferation.

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