Human type III recombinant collagen-related peptides and their applications

Abstract

This invention relates to the field of biotechnology, and in particular to human type III recombinant collagen-related peptides and their applications. This invention obtains human type III recombinant collagen-related peptides suitable for recombinant expression by selecting specific functional fragments from human type III collagen and repetitively constructing them. These peptides can be prepared using nucleic acids, recombinant vectors, and host cell systems, and are characterized by clear origin, controllable sequence, well-defined preparation pathways, and a wide range of applications. The obtained peptides exhibit good expression and purification properties and can promote fibroblast adhesion, proliferation, migration, or differentiation, thus making them suitable as functional components in materials used in the biomedical, tissue engineering, or cosmetic fields.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to human type III recombinant collagen-related peptides and their applications. Background Technology

[0002] Type III collagen, an abundant structural protein in the human body, possesses unique biological functions. As a signaling molecule, it participates in regulating cell adhesion, proliferation, migration, and differentiation, thereby enhancing the mechanical strength of bones, tendons, cartilage, and skin, and playing a role in maintaining skin firmness. Furthermore, type III collagen exhibits excellent biocompatibility, degradability, and low immunogenicity. For these reasons, type III collagen is widely favored in cosmetics, biomedicine, and tissue engineering.

[0003] In recent years, with the vigorous development of the social economy, the market demand for type III collagen has been continuously rising. Currently, animal-derived collagen is the main source of type III collagen, but its high extraction cost and the risks of immunogenicity, pathogen contamination, or allergic reactions limit its application. Therefore, obtaining highly active human type III recombinant collagen through biotechnology such as genetic engineering is attracting increasing attention. Summary of the Invention

[0004] The present invention aims to provide a human type III recombinant collagen-related polypeptide and its preparation and application methods. The polypeptide is constructed based on a specific amino acid sequence fragment from human type III collagen, which has the potential to form collagen-like structures and participate in extracellular matrix-related biological functions. By repeating this amino acid sequence once or multiple times, a human type III recombinant collagen-related polypeptide with good expression adaptability and biological activity can be obtained. This polypeptide can serve as a recombinant expression form of a functional fragment of human type III collagen, improving upon the shortcomings of naturally derived collagen in terms of source stability, batch-to-batch consistency, potential immunogenicity, pathogen contamination risk, and large-scale preparation.

[0005] The human type III recombinant collagen-related polypeptide provided by this invention is obtained by repeating the amino acid sequence shown in SEQ ID NO: 1 1 to 10 times. By setting the number of repetitions, the size of the polypeptide molecule, the content of collagen-like repeat units, the structural characteristics of the expression product, and its effect on fibroblast behavior can be adjusted while retaining the functional fragments related to human type III collagen.

[0006] In some preferred embodiments, the human type III recombinant collagen-related polypeptide is obtained by repeating the amino acid sequence shown in SEQ ID NO: 1 four times. This repetitive form is beneficial for balancing recombinant expression, isolation and purification, and cellular functional activity, demonstrating a good biological basis in experiments related to fibroblast adhesion, proliferation, migration, or differentiation.

[0007] The present invention also provides a nucleic acid encoding the aforementioned human type III recombinant collagen-related polypeptide. The nucleic acid may be codon-optimized according to the expression preferences of the host cell, and may also contain a nucleotide sequence or a repeat thereof corresponding to the amino acid sequence shown in SEQ ID NO: 1.

[0008] In some embodiments, the nucleic acid sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2. The nucleic acid can be used to construct a recombinant expression system to achieve stable expression of the human type III recombinant collagen-related polypeptide.

[0009] The present invention also provides a recombinant vector comprising the nucleic acid. The nucleic acid can be operatively linked to a promoter, enabling it to be transcribed and expressed as a target polypeptide in a suitable host cell.

[0010] In some embodiments, the recombinant vector may further include one or more elements that facilitate expression, screening, secretion, localization, stability, or purification, such as a promoter, signal peptide coding sequence, leader sequence coding sequence, terminator, marker gene, polyadenylated sequence, or tag sequence.

[0011] In some preferred embodiments, the tag sequence may be a 6×His tag to facilitate the separation and purification of the expression product by means of affinity chromatography or other methods.

[0012] The present invention also provides a host cell comprising the recombinant vector. The host cell may be a prokaryotic cell or a eukaryotic cell suitable for expressing the human type III recombinant collagen-related polypeptide.

[0013] In some embodiments, the host cell is a prokaryotic cell.

[0014] In some preferred embodiments, the host cell is Escherichia coli.

[0015] By introducing the nucleic acid encoding the polypeptide into host cells, engineered cells capable of expressing the human type III recombinant collagen-related polypeptide can be obtained, providing a foundation for subsequent fermentation culture, induced expression, and large-scale preparation.

[0016] The present invention also provides a method for preparing human type III recombinant collagen-related peptides.

[0017] The method includes culturing host cells containing the corresponding recombinant vector, inducing expression of the target protein under suitable conditions, and separating and purifying the expression product to obtain the human type III recombinant collagen-related polypeptide. In some embodiments, host cells can be amplified using a culture system with antibiotic selection, and the target polypeptide expression can be induced by an inducer; after expression, the bacterial cells or cells can be disrupted, clarified, and purified, and the target product can be obtained by affinity chromatography, molecular sieve chromatography, desalting, buffer replacement, or lyophilization. This method is beneficial for improving the controllability of the source and the consistency of preparation of human type III collagen-related polypeptides.

[0018] This invention also provides the application of the aforementioned human type III recombinant collagen-related peptide in promoting fibroblast adhesion, proliferation, migration, or differentiation. The peptide can provide a suitable extracellular matrix-like microenvironment for fibroblasts, promote cell-substrate interactions, and influence cell proliferation activity, scratch-induced migration ability, and the expression of differentiation-related markers. In some embodiments, these effects can be evaluated by cell adhesion rate, cell proliferation activity, cell migration rate, and the expression level of differentiation markers.

[0019] This invention also provides the application of the aforementioned human type III recombinant collagen-related peptide in the preparation of materials for use in the biomedical, tissue engineering, or cosmetic fields. The materials may be raw materials, compositions, coatings, scaffolds, dressings, gels, lyophilized powders, cell culture substrates, skin care materials, or tissue repair-related materials containing the peptide. In some embodiments, the peptide can improve the compatibility and functional cooperation between materials and cells through its regulatory effects on fibroblast adhesion, proliferation, migration, or differentiation, thereby providing functional protein components for tissue repair, cell culture, tissue engineering construction, and skin care-related products.

[0020] This invention obtains recombinant human type III collagen-related peptides suitable for recombinant expression by selecting specific functional fragments from human type III collagen and repetitively constructing them. These peptides can be prepared using nucleic acids, recombinant vectors, and host cell systems, and are characterized by their clear origin, controllable sequence, well-defined preparation pathway, and wide range of applications. In some embodiments, the obtained peptides exhibit good expression and purification properties and can promote fibroblast adhesion, proliferation, migration, or differentiation, thus making them suitable as functional components in materials used in the biomedical, tissue engineering, or cosmetic fields. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of plasmid construction for human type III recombinant collagen in an embodiment of the present invention; Figure 2 This is a schematic diagram of PCR gel electrophoresis of human type III recombinant collagen in an embodiment of the present invention; Figure 3 This is an SDS-PAGE gel image of human type III recombinant collagen in an embodiment of the present invention; Figure 4 This is an SDS-PAGE gel image of the stability of human type III recombinant collagen in an embodiment of the present invention. Figure 5 This is a circular dichroism spectrum of human type III recombinant collagen in an embodiment of the present invention; Figure 6 This is a graph showing the ultracentrifugation analysis (AUC) results of human type III recombinant collagen in an embodiment of the present invention; Figure 7 The image shows a fluorescence image of human type III recombinant collagen promoting cell adhesion in mouse embryonic fibroblasts in an embodiment of the present invention. The positive control is commercially available human type III collagen (purchased from Viimei), and the negative control is D-PBS. Figure 8 This is a graph showing the cell adhesion promotion rate of human type III recombinant collagen on mouse embryonic fibroblasts in an embodiment of the present invention; Figure 9 The results of the detection of cell proliferation activity of human type III recombinant collagen on mouse embryonic fibroblasts in the embodiments of the present invention are shown. The positive control is commercially available human type III collagen (purchased from Viimei), and the negative control is PBS. Figure 10 The image shows a microscopic image of the cell migration-promoting effect of human type III recombinant collagen on mouse embryonic fibroblasts in an embodiment of the present invention. The positive control is commercially available human type III collagen (purchased from Viimei), and the negative control is PBS. Figure 11 The figure shows the cell migration rate of mouse embryonic fibroblasts promoted by human type III recombinant collagen in an embodiment of the present invention. The positive control is commercially available human type III collagen (purchased from Viimei), and the negative control is PBS.

[0023] Figure 12 The figure shows the results of promoting cell differentiation of mouse embryonic fibroblasts with human type III recombinant collagen in an embodiment of the present invention. The positive control is commercially available human type III collagen (purchased from Viimei), and the negative control is D-PBS.

[0024] Figure 13 This is an SDS-PAGE gel image showing the expression of recombinant type III collagen in the comparative example of this invention. Detailed Implementation

[0025] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0026] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) should be understood as having the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of protection of this invention. Unless the context clearly defines otherwise, the scientific and technical terms used herein, as well as terms and laboratory procedures in interdisciplinary fields such as molecular biology, genetic engineering, protein engineering, and cell biology and biomaterials applications, are all conventional terms and standard methods well-known and widely used in the art. To facilitate understanding of the technical solutions of this invention, some related terms are further defined and explained below.

[0027] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0028] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0029] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0030] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted.

[0031] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0032] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity greater than or equal to 2.

[0033] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0034] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0035] In this invention, the term "polypeptide" refers to a linear or nonlinear molecule formed by two or more amino acids linked by peptide bonds, which can be an amino acid sequence obtained from natural sources, chemical synthesis, or recombinant expression.

[0036] In this invention, the term "human type III recombinant collagen-related polypeptide" refers to a polypeptide obtained by artificial design and recombinant expression based on an amino acid sequence fragment of human type III collagen.

[0037] In this invention, the term "repetition" refers to the process of tandemly arranging specific amino acid sequence fragments in the same or substantially the same order to form a continuous polypeptide structure. This repetition can be direct or indirect, and may also include the insertion of linker sequences between repeating units that do not affect the overall structure and function. Functionally, this repetition can be used to regulate the length, conformational characteristics, and cellular interaction of the polypeptide. Operationally, the corresponding nucleic acid sequence can be constructed using molecular cloning or chemical synthesis methods, and its expression status and conformational characteristics can be verified using protein expression and structural analysis techniques (e.g., SDS-PAGE, electrophoretic mobility, or circular dichroism spectroscopy).

[0038] In this invention, the term "nucleic acid" refers to a nucleotide molecule capable of encoding a polypeptide, including modified or unmodified DNA or RNA forms, as well as its complementary sequence, antisense sequence, or codon-optimized variants. The terms "nucleic acid molecule," "polynucleotide sequence," and "nucleotide sequence" are used interchangeably in this specification. This nucleic acid can functionally drive the expression of a target polypeptide in a host cell. Operationally, its sequence correctness can be verified by sequencing, and its expression capacity can be evaluated by detecting transcriptional or translational levels (e.g., mRNA expression levels or target protein expression levels). As used in this invention, nucleic acid includes, as a non-limiting example, all nucleic acid sequences obtained by any means available in the art, including, as a non-limiting example, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using common cloning techniques and PCR, etc., as well as synthetic means. Unless otherwise stated, the term "nucleotide sequence" includes its complement. Thus, a nucleic acid having a specific sequence should be understood to include a nucleic acid having its complementary strand. "Complementary" should be understood as the ability of the two to hybridize under stringent conditions. As used herein, the term “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the similarity between the two sequences is at least 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, more preferably 95%.

[0039] In this invention, the term "recombinant vector" refers to a nucleic acid construct containing the aforementioned nucleic acid and capable of replicating or expressing it in a host cell, which may include plasmids, viral vectors or other expression system elements.

[0040] In this invention, the term "host cell" refers to a cell capable of taking up and expressing the nucleic acid or recombinant vector, which may be a prokaryotic cell or a eukaryotic cell.

[0041] The first aspect of the present invention relates to a human type III recombinant collagen-related polypeptide, which is obtained by repeating the amino acid sequence shown in SEQ ID NO: 1 1 to 10 times.

[0042] In some embodiments, it is obtained by repeating the amino acid sequence shown in SEQ ID NO: 1 2, 3, 4, 5, 6, 7, 8, or 9 times; preferably 4 times.

[0043] As is generally understood by those skilled in the art, the concept of "human type III recombinant collagen-related polypeptide" also encompasses moderate variations thereof while retaining the activity of human type III recombinant collagen. Variations of the protein or polypeptide of this invention include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-10, preferably 1-5, more preferably 1-2, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. Variations of the polypeptide also include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, etc.

[0044] The present invention also provides a nucleic acid encoding a human type III recombinant collagen-related polypeptide as described above.

[0045] The nucleic acid may contain a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1, or a coding sequence formed by repeating and tandemly binding the nucleotide sequence, thereby enabling the expression of a polypeptide structure composed of repeating the amino acid sequence. The nucleic acid may be in the form of DNA or RNA, may be single-stranded or double-stranded, or may be a sequence variant codon-optimized to adapt to a specific host expression system. In some embodiments, the nucleic acid sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 is as shown in SEQ ID NO: 2.

[0046] The present invention also relates to a recombinant vector comprising the nucleic acid molecule described above, wherein the nucleic acid molecule is operatively linked to a promoter to drive expression.

[0047] The recombinant vector can be a plasmid vector or other expression vector form suitable for replication and expression in host cells. The vector can be introduced into host cells through transformation, transduction, or transfection, allowing its carried genetic material elements to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40), and insect baculoviruses. In some embodiments, the recombinant vector can be constructed based on a prokaryotic expression system, for example, by modifying pET series vectors. In some preferred embodiments, the recombinant vector can be a pET-32M-3C vector, which contains a T7 promoter and corresponding regulatory elements to achieve induced expression of the target polypeptide in *E. coli*. The nucleic acid molecule can be inserted into the multiple cloning site of the vector via restriction endonuclease recognition sites (e.g., BamHI and XhoI sites), thereby forming an operable link with the promoter. In some embodiments, this linking allows the target nucleic acid to be transcribed and translated into the target polypeptide under IPTG induction conditions. The expression function of the recombinant vector can be evaluated by detecting the expression level of the target protein after induction, electrophoretic bands, and the acquisition of purified products.

[0048] In some embodiments, the recombinant vector may further comprise one or more functional elements that facilitate the expression, stability, or purification of the target peptide. These elements comprise one or more selected from the group consisting of: i) Tag sequence; ii) Signal peptide coding sequence; iii) Preamble sequence encoding sequence; iv) Termination of contract; v) Marker genes; vi) Polyadenylated sequence.

[0049] In some preferred embodiments, the tag sequence can be a 6×His tag to facilitate the separation and purification of the target polypeptide by metal affinity chromatography. In some embodiments, the vector may also contain a Trx tag or a similar solubilizing tag to improve the soluble expression capacity of the target polypeptide and reduce inclusion body formation. In some embodiments, the terminator can be a T7 terminator to terminate the transcription process and improve expression stability. The marker gene can be an antibiotic resistance gene, such as an ampicillin resistance gene, used to screen successfully transformed host cells. The origin of replication (ori) can be used to maintain stable replication of the vector in the host cell. In some embodiments, the vector may also contain a lacI regulatory element to regulate the T7 promoter, thereby initiating target gene expression in the presence of an inducer. The above elements can exist individually or in combination to form a recombinant vector system suitable for target polypeptide expression, and its performance can be evaluated by indicators such as expression level, protein solubility, and purification efficiency.

[0050] The present invention also relates to host cells comprising the recombinant vector described above. The host cell may be a higher eukaryotic cell, such as an insect cell or a mammalian cell; a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell.

[0051] In some embodiments, the host cell is a prokaryotic cell, preferably Escherichia coli; more preferably Escherichia coli BL21(DE3).

[0052] In this invention, a method for preparing human type III recombinant collagen-related polypeptides is also provided, which includes culturing host cells as described above, inducing expression of the target protein, and separating and purifying the expression product.

[0053] During the culture process, a conventional culture medium system can be used. After reaching the logarithmic growth phase, expression can be induced. In some implementations, the expression of the target protein can be initiated by adding an inducer, and the expression level and protein solubility can be improved by adjusting the culture temperature, time and induction conditions.

[0054] In some embodiments, after expression, if desired, the target peptide can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0055] In some embodiments, the peptide can be purified using affinity chromatography, molecular sieve chromatography, or other protein purification methods, such as metal ion affinity chromatography using a tag sequence, to improve the purity of the target product. In further embodiments, the purified product can be subjected to buffer replacement or desalting to obtain a peptide formulation suitable for subsequent applications.

[0056] In some embodiments, the preparation method may further include evaluating the stability or structural characteristics of the expression product, such as analyzing its molecular weight and purity by electrophoresis and analyzing its secondary structure characteristics by spectroscopy, thereby confirming that the polypeptide has the expected structural basis. In some embodiments, its biological function may also be evaluated by combining cell experiments, such as detecting the adhesion rate, proliferation activity, or migration ability of fibroblasts, thereby further verifying the functional performance of the product obtained by the preparation method. The steps can be adjusted or combined according to specific needs to obtain human type III recombinant collagen-related polypeptides with good expression performance, purity, and biological activity.

[0057] This invention also provides the application of the aforementioned human type III recombinant collagen-related peptide in promoting fibroblast adhesion, proliferation, migration, or differentiation. The peptide can interact with fibroblast surface receptors or the extracellular environment by mimicking structural units or functional fragments in the extracellular matrix, thereby influencing cell attachment behavior, cytoskeleton remodeling, and signal transduction processes. In some embodiments, the effects can manifest as improving cell adhesion to substrate materials, enhancing cell proliferation, promoting cell migration in injury or scratch models, or regulating cell differentiation towards a specific phenotype.

[0058] In some embodiments, the polypeptide can exert its biological effects by contacting cells in solution, coated form, or as a material component. In further embodiments, the polypeptide can be used alone or in combination with other bioactive factors, matrix materials, or cell culture systems to enhance or modulate its influence on fibroblast behavior. The application can be evaluated using one or more detectable indicators, such as measuring cell adhesion rate using cell adhesion assays, detecting cell proliferation activity using CCK-8 or similar methods, evaluating cell migration ability using scratch assays or migration assays, and evaluating cell differentiation status by detecting the expression levels of differentiation-related markers (e.g., α-SMA, Vimentin, or Cadherin-related markers). These various effects can occur individually or in combination, demonstrating the application value of the human type III recombinant collagen-related polypeptide in regulating fibroblast behavior.

[0059] This invention also provides the application of the aforementioned human type III recombinant collagen-related peptide in the preparation of materials for use in the biomedical, tissue engineering, or cosmetic fields. The peptide can be introduced as a functional protein component into different types of material systems, improving the cell compatibility and functional performance of the materials by providing an extracellular matrix-like structure or regulating cell behavior. In some embodiments, the peptide can exist alone or in combination with other biomaterials or active ingredients to form composite materials with specific properties.

[0060] In some embodiments, within the biomedical field, the material can be a product related to tissue repair or regeneration, such as wound repair dressings, medical dressings, hemostatic materials, absorbable medical fillers, tissue repair support materials, or pharmaceutical excipients; the material can also be a carrier system for local drug delivery or bioactivity modulation, such as a protein carrier, a sustained-release system, or a local delivery matrix. In a further embodiment, the polypeptide can enhance the functional performance of the above-mentioned material in the tissue repair process by promoting fibroblast adhesion, proliferation, or migration.

[0061] In some embodiments, within the field of tissue engineering, the material can be a scaffold or matrix system for cell culture or tissue construction, such as a three-dimensional porous scaffold, hydrogel, nanofiber scaffold, thin film material, or cell culture substrate; the material can be used for in vitro cell expansion, tissue construction, or in vivo implantation-related applications. In some embodiments, the polypeptide can serve as an active component in the scaffold material, improving cell distribution, colonization, and function within the scaffold by regulating cell adhesion and migration behavior.

[0062] In some embodiments, within the cosmetic field, the material may be a functional ingredient or composition used in skin care or beauty-related products, such as serums, creams, lotions, masks, freeze-dried powder formulations, or skin repair compositions; the peptide may participate in the maintenance or improvement of skin structure through its regulatory effect on fibroblast behavior. In some embodiments, the peptide may be used in combination with other moisturizers, antioxidants, or active ingredients to form a synergistic cosmetic composition.

[0063] The aforementioned materials can be designed and adjusted according to specific application requirements. The polypeptides can be introduced into the material system in the form of solution, coating, blending or cross-linking. Their effects can be evaluated through cell compatibility experiments, cell function tests or related physicochemical indicators, thereby demonstrating their value in different application fields.

[0064] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these embodiments are only used to illustrate the technical content of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the specific experimental conditions in the following embodiments are given priority reference to the guidelines provided in this specification, or may be carried out according to generally accepted experimental manuals or conventional experimental conditions, or other experimental methods known in the art, or according to the conditions recommended by the relevant reagent or instrument manufacturers. In specific embodiments, unless otherwise specified, minor deviations within the weighing accuracy range are allowed for the measurement parameters involving raw material components; reasonable deviations due to instrument detection accuracy or operational accuracy are also allowed for parameters such as temperature and time.

[0065] Example 1: Construction of an Escherichia coli genetically engineered strain that efficiently expresses human type III recombinant collagen This invention provides a sequence with high biological activity potential, wherein the sequence is obtained by tandemly repeating an optimized high-activity fragment four times.

[0066] The amino acid sequence of the repeating unit is as shown in SEQ ID NO: 1: GPTGPQGPPGVKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPP The codon-optimized nucleotide sequence corresponding to SEQ ID NO: 1, suitable for expression in E. coli, is shown in SEQ ID NO: 2: GGCCCGACCGGCCCGCAGGGCCCGCCGGGCGTGAAGGGCAGCCCGGGCGAGCGTGGCGAAACCGGCCCGCCGGGCCCGGCGGGTTTTCCGGGCGCCCCGGGCCAAAACGGTGAACCGGGTGGCAAAGGCGAACGCGGTGCCCCGGGTGAGAAAGGCGAGGGTGGCCCACCGGGCGTTGCGGGCCCGCCGGGCGGCAGCGGTCCGGCGGGCCCGCCG The amino acid sequence obtained after four repeated tandem sequences is shown in SEQ ID NO: 3: SEQ ID NO: 3: GPTGPQGPPGVKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPTGPQGPPGVKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPP GPTGPQGPPGVKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPTGPQGPPGVKGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPP BamHI and XhoI restriction sites were added to the 5' and 3' ends of the nucleic acid sequence corresponding to SEQ ID NO: 3, respectively, and the gene was cloned into the vector PET-32M-3C (Ampicillin) via 5' BamHI and 3' XhoI to form a recombinant plasmid; wherein: Figure 1 This diagram shows the construct of a prokaryotic vector in *E. coli* to optimize the sequence. The vector contains a 6His tag for easier subsequent purification; a Trx tag, a solubilizing tag that enhances the solubility and stability of the target protein; a robust T7 promoter, an inducible promoter that allows control of protein expression by adding IPTG as an inducer; and an ampicillin resistance gene for screening successfully transformed strains.

[0067] The recombinant plasmid obtained above was verified by PCR, such as... Figure 2 As shown in the table below, the verification was successful, and the recombinant plasmid was successfully obtained. The PCR system is shown in the table below:

[0068] The recombinant plasmid was then transferred into Escherichia coli BL21(DE3) competent cells using a heat shock method to obtain an Escherichia coli genetically engineered strain that can efficiently express human type III recombinant collagen.

[0069] Example 2: Preparation and Validation of Human Type III Recombinant Collagen ① The strain obtained in Example 1 was picked and cultured in 1L of LB liquid medium containing Ampicillin resistance until OD 600Once the protein concentration reaches the range of 0.6 to 0.8, IPTG induction solution is added, and the cells are cultured at 16°C to induce protein expression. The cells are then centrifuged at 8000 rpm and 4°C to collect the bacteria. An appropriate amount of binding buffer is added to the collected cells, which are then resuspended and mixed with PMSF using a high-pressure homogenizer. The supernatant and precipitate are collected by centrifugation at 8000 rpm and 4°C, filtered through a 0.22 μm filter, and the supernatant is added to a nickel column. The column is bound at low temperature for 30 min. The nickel column is fixed on an iron stand, and the top and bottom seals are opened to collect the eluent. An appropriate amount of rinsing buffer is added, and the column is rinsed four times to remove any unbound protein. Samples of the rinsing solutions from the first two operations are taken. An appropriate amount of elution buffer is added to fully bind the eluent, and all the eluent is collected. 2.5 ml of the eluent is added to a desalting column. After all the sample has flowed out from the bottom, 6 ml of PBS buffer is added to displace the sample into the PBS solution. 4-5 ml of the eluent are collected. After the effluent is eluted, the desalting column is equilibrated, and the above operation is repeated until all samples are desalted to obtain human type III recombinant collagen.

[0070] ② The collected precipitate, supernatant, eluent, wash buffer, and desalting solution were prepared and subjected to SDS-PAGE electrophoresis at 120 V and 200 mA to verify protein expression. Figure 3 As shown, the obtained protein is a soluble protein with high expression level and high purity.

[0071] ③ Take a small amount of purified sample and place it at 4 ℃ and room temperature (25 ℃) for one week, respectively. Samples are taken at the same time points on days 1, 3, 5, and 7. Finally, SDS-PAGE electrophoresis is used to verify the protein's stability. Figure 4 As shown, the obtained protein exhibits good stability at both 4℃ and room temperature.

[0072] Example 3: Biochemical Analysis of Human Type III Recombinant Collagen ① Circular dichroism spectroscopy analysis The secondary structure of the protein was analyzed using a MOS-450 circular dichroism spectrometer. The scanning wavelength range was set to 180-260 nm, the scan rate to 50 nm / min, the temperature to 25 ℃, the scanning mode to continuous scan, and the bandwidth to 1 nm. The sample was diluted to 0.1 mg / ml with PBS buffer, and the protein sample was loaded into a 1 mm quartz cuvette for circular dichroism spectroscopy analysis. The detection data were then used to plot curves using Origin. Figure 5As shown, there are two negative peaks at wavelengths of 208 nm and 220 nm, and one obvious positive peak at wavelength of 193 nm. These are typical features of the α-helix structure, indicating that the purified protein sample has an α-helix structure, and it is speculated that it may have a spatial structure similar to that of natural proteins.

[0073] ② Ultracentrifugation analysis (AUC) Sedimentation rate analysis was performed using a Beckman Optima XL-I ultracentrifuge with an An-60 Ti rotor at 50,000 r / min and 4 ℃. Purified protein samples were diluted to 1 mg / ml with buffer, and a protein-free buffer was used as a control. The SV-AUC data were processed using Sedfit software. Figure 6 As shown, the obtained protein sample has a uniform conformation and high purity.

[0074] Example 4: Application of human type III recombinant collagen in helper fibroblasts ① Cell adhesion (1) Coating preparation: 100 μL of commercially available human III collagen (positive control, purchased from Viimei, 1.0 mg / mL), protein sample solution, and D-PBS negative control were added to each well of a 96-well plate. Three wells were prepared for each sample coating. The plates were incubated in an incubator for 1 h to 4 h. After that, the excess coating solution was removed from the wells, 100 μL of 1% BSA-PBS solution was added, and the plates were incubated in an incubator for 1 h. The liquid in the wells was then removed, and the plates were washed three times with D-PBS. The washing solution was discarded, and the plates were sealed with sealing film and stored at 4 ℃ for later use.

[0075] (2) Cell preparation and seeding: NIH / 3T3 cells (mouse embryonic fibroblasts) were cultured in an incubator. Cell density and status were observed daily. When the cells in the culture flasks reached 80%~90% confluence, cell seeding or passage was performed. The cells were diluted to 5×10⁻⁶ using complete culture medium mixed with Hoechst 33342 fluorescent staining agent (10%). 4 / mL. Add 100 μL of cells to the well, cover with aluminum foil, and incubate at 37 ℃, 5% for 1 h. Measure two replicate samples; the third well is used to adjust microscope parameters and its measurements are not applicable.

[0076] (3) Detection: Wash three times with PBS to remove unadhered cells. Detect the cell count in the 96-well plate using the CCK-8 assay and photograph the cells under a microscope. Figure 7 As shown, the number of fluorescent dots in the image was counted using ImageJ software, and the relative cell adhesion rate was calculated. Figure 8 As shown, its calculation formula is given in Equation 1: Relative cell adhesion rate (%) = Number of fluorescent spots in experimental group / Number of fluorescent spots in positive control group × 100% (Equation 1) Figure 8 This indicates that the cell adhesion-promoting effect of the human type III recombinant collagen sample prepared in this invention is not significantly different from that of the positive control (p ≥ 0.05), but the height of the bar chart is slightly higher than that of the positive control.

[0077] ②Cell proliferation (1) Add 100 μL of NIH / 3T3 cell (mouse embryonic fibroblast) suspension to a 96-well plate, place the culture plate in an incubator, and pre-culture for 24 h at 37 ℃ and 5% CO2. (2) Add 10 μL (0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml) of the test sample to the culture plate, and use commercially available human type III collagen (purchased from Viimei, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml) as a positive control. Incubate the culture plate in an incubator for 6 h, 12 h, 24 h and 48 h respectively. (3) Add 10 μL of CCK-8 solution to each well (note that air bubbles should be avoided during the sample addition process, otherwise it will affect the reading of OD value), and place the culture plate in the incubator to continue incubation for 2 h (to ensure sufficient color development); (4) Measure the absorbance at 450 nm using an ELISA reader and calculate the cell proliferation activity, such as... Figure 9 As shown, the calculation formula is given in Equation 2: Cell proliferation activity (%) = [ A (Add sample to be tested) - A (blank)] / [ A (0 plus test sample) - A [(Blank)] × 100% (Equation 2) in, A (with sample to be tested): absorbance of the well containing cells, CCK-8 solution and the sample solution; A (Blank): Absorbance of pores containing culture medium and CCK-8 solution but without cells; A (0 plus sample): Absorbance of wells containing cells and CCK-8 solution but no sample solution.

[0078] Figure 9The results show that the human type III recombinant collagen prepared in this invention showed no significant difference compared with the positive control at a culture time of 12 h and a concentration of 0.1 mg / ml and 0.5 mg / ml (p ≥ 0.05); however, at a culture time of 12 h and a concentration of 1 mg / ml and 2 mg / ml, the cell proliferation activity of the human type III recombinant collagen prepared in this invention (p < 0.05) was significantly higher than that of the positive control.

[0079] ③ Cell migration (1) NIH / 3T3 cell (mouse embryonic fibroblast) culture: Mark each well of a 6-well plate with a marker pen, dividing each well into three equal parts horizontally and vertically, approximately 5 × 10⁶ cells per well. 5 Individual cells are seeded and cultured for 24 hours until they reach 95%-100% confluence. (2) Scratch test: Use a 10 μL pipette tip to directly aim at the well plate and gently push downwards to form a longitudinal scratch. Wash the cells with PBS 3 times to remove the scratched cells. Add 2 mL of serum-free culture medium to the sample group (human type III recombinant collagen prepared in this invention, concentration 0.5 mg / mL) and the positive control group (commercially available human type III collagen, purchased from Viimei, concentration 0.5 mg / mL) to the test sample and control material, with a final concentration of 0.5 mg / mL. The blank control group only added serum-free culture medium. Each group had 3 replicate wells. (3) Culture and observation: Cultured in an incubator, at 0 h and 24 h, photographs were taken under a 40x microscope, using the intersection of the horizontal and vertical streaks as the core, and the images were saved. Figure 10 As shown; Data processing: ImageJ software was used to measure the migration area of ​​the scratched region in the image and calculate the cell migration rate, such as... Figure 11 As shown, the calculation formula is given in Equation 3: Cell migration rate (%) = (migration area of ​​scratch region / initial area of ​​scratch region) × 100% (Equation 3) Figure 11 The results showed that, compared with the positive control, the cell migration-promoting ability of the human type III recombinant collagen sample prepared in this invention was significantly higher than that of the positive control (p < 0.0001).

[0080] ④ Cell differentiation (1) Coating preparation: The sidewalls of the 6-well plate were marked with a marker pen to indicate the sample group, positive control group and negative control group. 2 mL of 5 mg / mL sample solution (human type III recombinant collagen sample prepared in this invention, concentration 0.5 mg / mL), positive control (commercially available human type III collagen, purchased from Viimei, concentration 0.5 mg / mL) and D-PBS negative control were added to the 6-well plate respectively. After incubation in an incubator for 1-4 hours, the excess coating solution in the well plate was discarded. 2 mL of 1% BSA-PBS blocking solution was added to the well. After incubation in an incubator for 1 hour, the blocking solution in the well was discarded. After washing three times with D-PBS, the washing solution was discarded. The well plate was sealed with sealing film and placed in a refrigerator at 4°C for later use.

[0081] (2) Cell seeding and culture: 5 × 10⁶ cells were seeded into each well of a 6-well plate with the prepared coating. 4 Target cells were seeded into each well at a density of 100 cells, then complete culture medium was added, and the cells were incubated in an incubator for 4 days before the cell differentiation was assessed.

[0082] (3) RT-PCR detection and data analysis: Cell samples were collected from each well, total RNA was extracted from the cells, reverse transcription was performed, cDNA was synthesized, and the cDNA was used as a template to perform real-time PCR reaction for cell differentiation markers (such as α-SMA, Vimentin, Cadherin-11) to detect the relative expression level of each marker gene. Commercially available human type III collagen (purchased from Viimei) was used as a positive control to compare and analyze the promoting effect of human type III recombinant collagen samples on cell differentiation.

[0083] Data processing: using 2 -ΔΔCt The relative expression levels of each marker are calculated using the following formula: ΔCt = Target gene Ct value - Internal reference gene Ct value (same sample) (Equation 4) ΔCt = experimental group ΔCt ΔCt value of the control group (Equation 5) Relative expression level = 2 -ΔΔCt (Equation 6) Figure 12The results showed that, compared with the negative control, the relative expression levels of the mesenchymal marker Vimentin and α-SMA in the human type III recombinant collagen sample were significantly increased (p<0.0001) and significantly increased (p<0.05). In contrast, the relative expression level of the epithelial marker Cadherin-1 in the negative control was significantly higher than that in the human type III recombinant collagen sample (p<0.001). Compared with the positive control, the relative expression level of the mesenchymal marker Vimentin in the human type III recombinant collagen sample prepared in this invention was significantly increased (p<0.01), while the relative expression level of α-SMA showed no significant difference (p ≥0.05). Furthermore, the relative expression level of the epithelial marker Cadherin-1 in the positive control was significantly higher than that in the human type III recombinant collagen sample (p<0.0001), indicating that the human type III recombinant collagen prepared in this invention has significant activity in inducing epithelial-mesenchymal phenotypic transformation.

[0084] Comparative Example The highly active fragment of human type III collagen obtained in this invention is based on bioinformatics. It is obtained by performing bioinformatics prediction on the physicochemical properties, hydrophilicity / hydrophobicity, spatial structure, and biological activity score of the full-length human type III collagen gene sequence, and selecting the fragment with the high activity score for optimization.

[0085] Under the experimental conditions of Example 1, the sequence shown in SEQ ID NO: 1 was replaced with SEQ ID NO: 4, and everything else was the same as in Example 1; SEQ ID NO: 4: GENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPP.

[0086] Following the conditions of Example 2, the obtained plasmid was transformed into E. coli and expression was induced. The results are as follows: Figure 13 As shown, the presence of inclusion bodies in the protein expression indicates that the optimized sequence SEQ ID NO.4 has certain defects. This suggests that there are still shortcomings in the recombinant collagen preparation technology in the stages of protein sequence screening and design, expression system construction, and industrial production. If the process technology is not mature enough, a series of problems such as unstable product quality, low production efficiency, and failure to meet bioactivity standards may occur.

[0087] This study prepared recombinant type III collagen. Multiple experimental results showed that the product exhibited high protein expression levels, good purification, high protein expression concentration, and good stability. Compared with commercially available human type III collagen, it demonstrated superior bioactivity, providing experimental support for large-scale industrial production. The recombinant type III collagen prepared in this invention can effectively act on fibroblasts, significantly improving and promoting the adhesion, growth, proliferation, directional migration, and differentiation abilities of NIH / 3T3 cells. It can be widely used in various fields such as skincare and cosmetic raw materials, tissue engineering scaffolds, medical consumables, and pharmaceutical excipients.

[0088] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A human type III recombinant collagen-related polypeptide, which is obtained by repeating the amino acid sequence shown in SEQ ID NO: 1 1 to 10 times.

2. The human type III recombinant collagen-related polypeptide according to claim 1, which is obtained by repeating the amino acid sequence shown in SEQ ID NO: 1 four times.

3. A nucleic acid encoding the human type III recombinant collagen-related polypeptide as described in claim 1 or 2.

4. The nucleic acid according to claim 3, wherein the nucleic acid sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO:

2.

5. A recombinant vector comprising the nucleic acid molecule of claim 3 or 4, wherein the nucleic acid molecule is operatively linked to a promoter to drive expression.

6. The recombinant vector according to claim 5, further comprising one or more elements selected from the group consisting of: i) Tag sequence, preferably 6×His tags; ii) Signal peptide coding sequence; iii) Preamble sequence encoding sequence; iv) Termination of contract; v) Marker genes; vi) Polyadenylated sequence.

7. A host cell comprising the recombinant vector of claim 5 or 6, preferably a prokaryotic cell, more preferably Escherichia coli.

8. A method for preparing human type III recombinant collagen-related peptides, comprising the following steps: The host cells described in claim 7 are cultured to induce the expression of the target protein, and the expression product is separated and purified.

9. The use of the human type III recombinant collagen-related polypeptide of claim 1 or 2 in promoting fibroblast adhesion, proliferation, migration or differentiation.

10. The use of the human type III recombinant collagen-related polypeptide of claim 1 or 2 in the preparation of materials for use in the fields of biomedicine, tissue engineering or cosmetics.

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