Extracellular matrix polymer protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to extracellular matrix polymer protein and a preparation method and application thereof. The invention also relates to a recombinant protein composition, a polynucleotide composition, a carrier composition, a recombinant cell composition and an active additive or preparation used in the fields of tissue engineering, pharmacy or beauty and skin care related to the extracellular matrix multimeric protein. The extracellular matrix polymer protein comprises a first subunit and a second subunit, the first subunit comprises a first coiled-coil domain and at least one extracellular matrix protein fragment; the second subunit comprises a second coiled-coil domain and at least one extracellular matrix protein fragment; the first subunit and the second subunit are non-covalently bound via the first coiled helical domain and the second coiled helical domain. The extracellular matrix polymer protein overcomes at least one of the defects of rigid multi-module assembly structure, insufficient activity retention and low yield after fusion of the traditional artificially prepared extracellular matrix protein.

Description

Technical Field

[0001] This application relates to the field of biotechnology, particularly to extracellular matrix multimeric proteins and their preparation methods and applications, as well as to recombinant protein compositions, polynucleotide compositions, carrier compositions, recombinant cell compositions, and active additives or formulations for use in tissue engineering, pharmaceutical or cosmetic fields related to extracellular matrix multimeric proteins. Background Technology

[0002] The extracellular matrix (ECM) is a network of macromolecular polymers secreted by cells, mainly composed of core proteins such as collagen, elastin, fibronectin, and laminin. It serves a dual function of structural support and bioactivity regulation. In skin tissue, the ECM not only provides mechanical support for epidermal cells and fibroblasts, maintaining skin elasticity and firmness, but also regulates cell proliferation, differentiation, and collagen synthesis by releasing signaling molecules, directly impacting key physiological processes such as skin aging and damage repair.

[0003] Due to their superior biocompatibility and ability to adhere to the skin's physiological structure, ECM proteins have become a core research and development direction for anti-aging and repair cosmetic ingredients. Currently, the preparation technologies for ECM proteins and their derivatives used in the cosmetics field are mainly divided into three categories, each with significant drawbacks:

[0004] (1) Early technologies used animal tissues as raw materials to obtain ECM protein fragments (such as collagen peptides and elastin hydrolysates) through acid hydrolysis, enzymatic hydrolysis, or chemical extraction. The advantage of this technology is that the raw materials are readily available, but it has significant limitations, including: low purity, easy contamination with allergenic impurities such as proteins and lipids, which does not meet the safety requirements of cosmetic raw materials; poor structural integrity, as enzymatic hydrolysis / chemical treatment will destroy the natural tertiary structure of ECM proteins, resulting in a significant decrease in biological activity (such as cell adhesion ability); and poor batch stability, as the molecular weight and activity of different batches of products vary significantly due to the influence of raw material species and breeding environment, making it difficult to standardize production.

[0005] (2) Short peptides containing ECM protein core sequences (such as RGD adhesion sequences) are prepared by solid-phase synthesis. However, this technology has two major problems: on the one hand, residual chemical reagents during the synthesis process may cause skin irritation; on the other hand, it also has the defects of weak assembly ability, lack of stable interaction between short peptide molecules, and inability to form the three-dimensional network structure of natural ECM.

[0006] (3) Microbial expression systems such as Escherichia coli and Pichia pastoris can be used to directionally clone core functional units of ECM and integrate multiple modules through direct gene fusion or covalent linking of flexible peptide links. However, this conventional covalent integration strategy has significant technical bottlenecks: First, biological activity may be impaired: directly and forcibly covalently fusing multiple protein modules with different functions (or using linkers to connect them) is essentially a "rigid splicing" of protein sequences. The spatial conformations of multiple modules are prone to mutual interference, leading to misfolding of the overall protein; the active sites of some modules may also be masked by adjacent sequences, which may eventually lead to the decline of core physiological functions of ECM proteins such as cell adhesion. Second, it will also lead to a decrease in expression yield: a single module can usually be expressed efficiently in a microbial chassis, but after multiple modules are fused into a complex protein, the length of the amino acid sequence and the complexity of the spatial structure increase sharply, which will increase the transcriptional and translational burden of the host cell, resulting in a decrease in the expression level of the target protein, making it difficult to meet the needs of industrial production.

[0007] Traditional techniques for the biosynthesis of ECMs revolve around "recombinant protein construction or fusion expression," but covalent fusion construction of ECMs faces a core technological bottleneck: it is difficult to simultaneously achieve "mild and stable assembly of multiple modules, preservation of bioactivity, and efficient expression of the microbial chassis." Simply mixing recombinant elastin, recombinant collagen, and recombinant fibronectin does not allow the components to spontaneously form the three-dimensional network structure of a natural ECM. Traditional fusion protein techniques either result in structural rigidity due to covalent assembly, leading to a loss of activity, or reduce yield due to the complexity of the fusion protein structure. Ultimately, the constructed ECM proteins fail to meet the industrial demands of "high activity and high yield" in the beauty industry.

[0008] In summary, the inherent defects of traditional technologies (rigid multi-module assembly structure, insufficient activity retention, and low yield after fusion) form a "triple contradiction," restricting the industrial upgrading of ECM proteins in the beauty industry. Summary of the Invention

[0009] Therefore, it is necessary to provide an extracellular matrix multimeric protein, its preparation method, and its application.

[0010] In order to achieve the above-mentioned objectives of this application, the following technical solution is adopted:

[0011] In a first aspect, an extracellular matrix multimer protein is provided, the extracellular matrix multimer protein comprising a first subunit and a second subunit;

[0012] The first subunit comprises a first coiled helical domain and at least one extracellular matrix protein fragment;

[0013] The second subunit comprises a second coiled-coil domain and at least one extracellular matrix protein fragment;

[0014] The first subunit and the second subunit are non-covalently bonded through a first coiled helical structural domain and a second coiled helical structural domain.

[0015] In an optional embodiment, the extracellular matrix protein fragments in the first subunit and the second subunit each independently comprise at least one of collagen fragments, elastin fragments, and fibronectin fragments.

[0016] In an optional embodiment, the collagen fragment includes a type III collagen fragment. Further optionally, the amino acid sequence of the type III collagen fragment includes the sequence shown in SEQ ID NO. 5, or includes a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO. 5.

[0017] In an optional embodiment, the amino acid sequence of the elastin fragment includes the sequence shown in SEQ ID NO.7, or includes a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.7.

[0018] In an optional embodiment, the amino acid sequence of the fibronectin fragment includes the sequence shown in SEQ ID NO.8, or includes a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.8.

[0019] In an optional embodiment, the extracellular matrix protein fragment in the first subunit includes the fibronectin fragment and the elastin fragment.

[0020] In an optional embodiment, the amino acid sequence of the extracellular matrix protein fragment in the first subunit includes the sequence shown in SEQ ID NO.2, or includes a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.2.

[0021] In an optional embodiment, the extracellular matrix protein fragment in the second subunit comprises the type III collagen fragment.

[0022] In an optional embodiment, the first coiled helical structural domain is located at the C-terminus of the first subunit.

[0023] In an optional embodiment, the second coiled helical structural domain is located at the C-terminus of the second subunit.

[0024] In an optional embodiment, the first and second coiled helical structural domains include P3S and P4S.

[0025] In an optional implementation, the first subunit and the second subunit are expressed independently by eukaryotic cells or prokaryotic cells, respectively.

[0026] In an optional implementation, the first subunit and the second subunit are expressed by Escherichia coli.

[0027] In an optional embodiment, the first subunit comprises, from the N-terminus to the C-terminus, a fibronectin fragment, an elastin fragment, and a coiled-coil domain P3S; the second subunit comprises, from the N-terminus to the C-terminus, a type III collagen fragment and a coiled-coil domain P4S.

[0028] In an optional embodiment, the amino acid sequence of the first subunit includes the sequence shown in SEQ ID NO.3, or includes a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO.3.

[0029] In an optional embodiment, the amino acid sequence of the second subunit includes the sequence shown in SEQ ID NO. 6, or includes a sequence having at least 80% sequence identity with the sequence shown in SEQ ID NO. 6.

[0030] In an optional embodiment, the extracellular matrix multimer protein is a heterodimer, the amino acid sequence of the first subunit includes the sequence shown in SEQ ID NO.3; the amino acid sequence of the second subunit includes the sequence shown in SEQ ID NO.6; the coiled-coil domain P3S and the coiled-coil domain P4S are non-covalently bound; both the first subunit and the second subunit are expressed by Escherichia coli.

[0031] In a second aspect, a recombinant protein composition is provided, the recombinant protein composition comprising a first subunit and a second subunit as defined in the first aspect, each separately packaged.

[0032] Thirdly, a polynucleotide composition is provided, the polynucleotide composition comprising a first polynucleotide and a second polynucleotide; the first polynucleotide encodes a first subunit as defined in the first aspect; and the second polynucleotide encodes a second subunit as defined in the first aspect.

[0033] Fourthly, a recombinant vector composition is provided, the recombinant vector composition comprising a first recombinant vector and a second recombinant vector; wherein the first recombinant vector carries a first polynucleotide as defined in the second aspect, and the second recombinant vector carries a second polynucleotide as defined in the second aspect.

[0034] Fifthly, a recombinant cell composition is provided, comprising a first recombinant cell and a second recombinant cell; wherein the first recombinant cell contains a first polynucleotide as defined in the second aspect, or contains a first vector as defined in the third aspect, or expresses a first subunit as defined in the first aspect;

[0035] The second recombinant cell contains the second polynucleotide as defined in the second aspect, or contains the second carrier as defined in the third aspect, or expresses the second subunit as defined in the first aspect.

[0036] In a sixth aspect, a method for preparing the extracellular matrix multimer protein described in the first aspect is provided, the method comprising mixing the first subunit and the second subunit, thereby causing the first subunit and the second subunit to spontaneously assemble into a multimer in a solvent.

[0037] In an optional embodiment, the freeze-dried first subunit and the freeze-dried second subunit are first mixed, and then the mixture is dissolved in a solvent.

[0038] In an optional embodiment, the mass ratio of the freeze-dried first subunit to the freeze-dried second subunit is 1:(2~3), and more preferably 1:2.3.

[0039] In an optional embodiment, the pH of the solvent is 3 to 4.

[0040] In an optional embodiment, the acid used to provide an acidic environment in the solvent includes at least one of acetic acid and citric acid.

[0041] In an optional embodiment, the solvent is a 0.1% v / v aqueous solution of acetic acid.

[0042] In an optional embodiment, the preparation method further includes culturing the first recombinant cell and the second recombinant cell as defined in the fifth aspect, respectively, and then separating and purifying the first subunit and the second subunit.

[0043] In a seventh aspect, a method for preparing the extracellular matrix multimer protein of the first aspect, or the recombinant protein composition of the second aspect, or the polynucleotide composition of the third aspect, or the recombinant carrier composition of the fourth aspect, or the recombinant cell composition of the fifth aspect, or the extracellular matrix multimer protein of the sixth aspect, is provided to obtain the application of the extracellular matrix multimer protein in the preparation of formulations for use in tissue engineering, pharmaceutical or cosmetic skin care fields.

[0044] Eighthly, an active additive or formulation for use in tissue engineering, pharmaceutical or cosmetic fields is provided, said active additive or formulation comprising the extracellular matrix multimer protein described in the first aspect, or the extracellular matrix multimer protein obtained by the preparation method described in the sixth aspect.

[0045] The extracellular matrix multimer protein provided in this application achieves multi-module assembly through non-covalent interactions mediated by coiled-coil domains in each subunit, which alleviates the module conformation interference caused by traditional covalent fusion, solves the assembly rigidity problem, and can also form a natural-like three-dimensional network structure.

[0046] The extracellular matrix multimer protein provided in this application is obtained through non-covalent interactions. These non-covalent interactions are gentle; that is, by adding short-chain protein-protein interaction pairing domains to the extracellular matrix protein fragments to be assembled, the two proteins can spontaneously assemble into stable non-covalent protein clusters in vitro. This maximizes the preservation of the native spatial conformation of each unit, ensuring the biological activity of the ECM protein. By fusing the core functional modules of the ECM with short coiled-coil domains, assembly is completed using gentle non-covalent interactions between the paired coiled-coil domains (rather than traditional covalent fusion). This fundamentally avoids the destruction of the natural spatial conformation of functional modules caused by direct splicing or linker tandem, effectively preserving their core biological activities such as cell adhesion. Simultaneously, each functional module can be independently and efficiently expressed in microorganisms before reassembly, avoiding the stress on the expression system caused by multi-module covalent fusion and significantly increasing yield. This "split-assemble" strategy avoids the burden on host cells from complex fusion proteins, increases the expression level of single modules, and meets industrialization requirements.

[0047] The coiled helix-mediated multi-module non-covalent assembly strategy has the advantages of "mild assembly" and "activity retention". At present, the application of coiled helix domains in the construction of cosmetic ECM proteins is still in the blank stage. This application applies it to the cosmetic scenario, which will help provide the cosmetic field with core raw materials with "high activity and high yield". Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0049] Figure 1 This is a schematic diagram illustrating the design of the sequences of the two non-covalently binding subunits in Example 1.

[0050] Figure 2This is a plasmid map of the expression plasmid for the recombinant rhEMp_P3S protein in Example 1;

[0051] Figure 3 This is a plasmid map of the expression plasmid for the recombinant ColⅢ_P4S protein in Example 1;

[0052] Figure 4 The results of SDS-PAGE gel electrophoresis of the concentrated supernatant samples of rhEMp_P3S and ColⅢ_P4S proteins in Example 1 and the results of SDS-PAGE gel electrophoresis of the ECM-SA protein prepared in Example 3 are shown.

[0053] Figure 5 The shake-flask yields of rhEMp_P3S, ColⅢ_P4S and rhECMp control proteins in Example 1;

[0054] Figure 6 This is a molecular structure diagram of the ECM-SA protein spontaneously assembled from rhEMp_P3S and ColⅢ_P4S in Example 3;

[0055] Figure 7 The cell adhesion rate of each protein in the cell adhesion experiment of Example 4 is shown. Detailed Implementation

[0056] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0058] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0059] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0060] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0061] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0062] In this application, "separately independent", "each...independently selected" and "...separately independently selected" and "...independently selected" are interchangeable and should be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0063] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0064] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0065] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0066] In this application, "coiled-coil domain" refers to a domain that can form homologous or heterologous polymers, such as dimers or trimers, through a coiled-coil structure.

[0067] In this application, "non-covalent bonding" refers to the bonding between molecules or between atoms within a molecule through non-covalent interactions. In the context of "extracellular matrix multimeric proteins," it refers to the formation of multimeric proteins between the first and second subunits through non-covalent interactions. Types of "non-covalent bonding" include, but are not limited to, hydrogen bonds, ionic bonds, van der Waals forces, coordination bonds, electrostatic interactions, or hydrophobic interactions. Multimers formed by coiled-helical domains typically utilize non-covalent bonding mechanisms including hydrophobic interactions, hydrogen bonds, and electrostatic interactions.

[0068] In this application, "extracellular matrix proteins" refers to proteins that constitute the extracellular matrix. Extracellular matrix proteins are the main components of the extracellular matrix and include various structural proteins, adhesion proteins, glycoproteins, and proteoglycans. Examples of extracellular matrix proteins include, but are not limited to, collagen (e.g., but not limited to type I collagen, type II collagen, type III collagen, and type VII collagen), fibronectin, or elastin. Collagen consists of three peptide chains forming a triple helix structure, possessing high tensile strength and providing mechanical support for tissues. Elastin imparts elasticity to tissues, enabling them to stretch and rebound. Elastin contains numerous proline and lysine residues, which cross-link to form an elastic network. Fibronectin is a large glycoprotein containing multiple functional domains. It can bind to cell surface receptors (such as integrins), connecting cells to the extracellular matrix. Fibronectin plays an important role in cell adhesion, migration, and differentiation. This application does not limit the species source of the extracellular matrix protein. In optional embodiments, the extracellular matrix protein is derived from mammals, including but not limited to any one of mice, rats, monkeys, chimpanzees, pigs, cattle, sheep, rabbits, horses, donkeys, deer, or camels. In some embodiments, the extracellular matrix protein is a human-derived extracellular matrix protein.

[0069] In this application, any protein fragment may be a polypeptide containing all or part of the amino acids of the protein or any subunit of the protein, and the amino acid sequence of any protein fragment may be derived from a natural protein or an artificially modified protein, including but not limited to mutation, insertion or truncation.

[0070] In this application, peptides, polypeptides, and proteins are not strictly distinguished and can be used interchangeably in some cases. Generally, peptides refer to polyamino acids linked by peptide bonds, and are not limited to being naturally occurring or synthetic. Polypeptides may also contain non-amino acid components as modifying groups, such as carbohydrate groups, metal ions, or carboxylic acid esters. Non-amino acid components may be added by the cells expressing the polypeptide and may vary depending on the cell type. Polypeptides are defined herein based on their amino acid backbone structure or the nucleic acid encoding them.

[0071] In this application, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a polynucleotide encodes a protein or polypeptide, it optionally encodes either the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0072] In this application, the term "vector" refers to a delivery vehicle that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) therein and enable the expression of that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. For example, vectors include: plasmids, episome plasmids, microcircular DNA, phage particles, cosmid 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, etc. Vectors may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into cells, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vectors (e.g., expression vectors) provided in this disclosure contain a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in this disclosure, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one selection marker.

[0073] In this application, the term "recombinant cell" refers to a cell into which exogenous polynucleotides and / or vectors can be or have been introduced. The exogenous polynucleotides may or may not be integrated into the genome of the "recombinant cell." Vectors can be introduced into the cell to construct recombinant cells, which can then be used to express a target protein. The recombinant cell can be cultured to obtain the corresponding target protein. Recombinant cells can be prokaryotic or eukaryotic cells. Prokaryotic cells include, but are not limited to, *Escherichia coli*, *Bacillus*, or *Staphylococcus*. Eukaryotic cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include, but are not limited to, yeast and filamentous fungal cells.

[0074] In one aspect, some embodiments provide an extracellular matrix multimer protein comprising a first subunit and a second subunit.

[0075] The first subunit comprises a first coiled-coil domain and at least one extracellular matrix protein fragment, and the second subunit comprises a second coiled-coil domain and at least one extracellular matrix protein fragment; and the first and second subunits are non-covalently bound through the first and second coiled-coil domains.

[0076] The extracellular matrix protein fragments in the first subunit and the second subunit may be the same or different. In an optional embodiment, the extracellular matrix protein fragments in the first subunit and the second subunit are different.

[0077] In an optional embodiment, the extracellular matrix protein fragment in the first subunit includes at least one of collagen fragments, elastin fragments, and fibronectin fragments.

[0078] In an optional embodiment, the extracellular matrix protein fragment in the second subunit includes at least one of collagen fragments, elastin fragments, and fibronectin fragments.

[0079] In an optional embodiment, the collagen fragment includes a type III collagen fragment. Further optionally, the amino acid sequence of the collagen fragment includes the sequence shown in SEQ ID NO. 5, or includes a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO. 5.

[0080] SEQ ID NO.5:

[0081] MGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGP NGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGF PGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGP NGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFP

[0082] In an optional embodiment, the amino acid sequence of the elastin fragment includes the sequence shown in SEQ ID NO.7, or includes a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO.7.

[0083] SEQ ID NO.7:

[0084] VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT

[0085] In an optional embodiment, the amino acid sequence of the fibronectin fragment includes the sequence shown in SEQ ID NO.8, or includes a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO.8.

[0086] SEQ ID NO.8:

[0087] VPGIGVPGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIGAVTGRGDSPASSVPGIGVPGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIG

[0088] In an optional embodiment, the extracellular matrix protein fragment in the first subunit comprises a fibronectin fragment and an elastin fragment. Further optionally, the amino acid sequence of the extracellular matrix protein fragment in the first subunit comprises the sequence shown in SEQ ID NO. 2, or comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO. 2.

[0089] SEQ ID NO.2:

[0090] MVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGGGSVPGIGV PGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIGAVTGRGDSPASSVPGIGVPGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIG

[0091] In an optional embodiment, the extracellular matrix protein fragment in the second subunit comprises a type III collagen fragment. Further optionally, the amino acid sequence of the extracellular matrix protein fragment in the second subunit comprises the sequence shown in SEQ ID NO. 5, or comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO. 5.

[0092] In an optional implementation, the first coiled helical structural domain is located at the C-terminus of the first subunit.

[0093] In an optional implementation, the second coiled helical structural domain is located at the C-terminus of the second subunit.

[0094] In an optional embodiment, the first and second coiled-coil domains include P3S and P4S, and there is no limitation that the first and second coiled-coil domains correspond to the coiled-coil domains in the combination, as long as they form peptide pairs that can bind non-covalently. The aforementioned coiled-coil can "specifically pair and bind," that is, when different extracellular matrix proteins each carry this "pairing fragment," they can precisely aggregate together to form a multi-protein complex, avoiding aimless aggregation between monomers.

[0095] The amino acid sequence of the coiled-helix domain P3S includes the sequence shown in SEQ ID NO.1, or the sequence showing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO.1.

[0096] SEQ ID NO.1:EIQQLEEEISQLEQKNSQLKEKNQQLKYG

[0097] The amino acid sequence of the coiled-coil domain P4S includes the sequence shown in SEQ ID NO.4, or includes a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO.4.

[0098] SEQ ID NO.4:KISQLKQKIQQLKQENQQLEEENSQLEYG

[0099] In an optional implementation, the first subunit and the second subunit are expressed independently by eukaryotic cells or prokaryotic cells, respectively.

[0100] In an optional implementation, the first and second subunits are expressed by Escherichia coli.

[0101] In an optional embodiment, the first subunit comprises, from the N-terminus to the C-terminus, a fibronectin fragment, an elastin fragment, and a coiled-coil domain P3S. Further optionally, the amino acid sequence of the first subunit includes the sequence shown in SEQ ID NO.3, or includes a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO.3.

[0102] SEQ ID NO.3:

[0103] MVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTGGGGSVPGIGVPGIGVPGKGVPGIG VPGIGVPGIGVPGIGVPGKGVPGIGVPGIGAVTGRGDSPASSVPGIGVPGIGVPGKGVPGIGVPGIGVPGIGVPGIGVPGKGVPGIGVPGIGEIQQLEEEISQLEQKNSQLKEKNQQLKYG

[0104] The second subunit comprises, from the N-terminus to the C-terminus, a type III collagen fragment and a coiled-coil domain P4S. Optionally, the amino acid sequence of the second subunit includes the sequence shown in SEQ ID NO. 6, or includes a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO. 6.

[0105] SEQ ID NO.6:

[0106] MGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEK GPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGF RGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFP GAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPGAPGERGERGAPGFRGPAGPNGIPGEKGPAGERGAPGVMGFPKISQLKQKIQQLKQENQQLEEENSQLEYG

[0107] In a further optional embodiment, the extracellular matrix multimer protein is a heterodimer, the amino acid sequence of the first subunit includes the sequence shown in SEQ ID NO.3; the amino acid sequence of the second subunit includes the sequence shown in SEQ ID NO.6; the coiled-coil domain P3S of the first subunit and the coiled-coil domain P4S of the second subunit are non-covalently bound to form a heterodimer; both the first and second subunits are expressed by Escherichia coli.

[0108] This implementation method utilizes a construction pathway of "independent expression of functional modules + coiled-coil domain-mediated assembly" to decompose the ECM protein into two-module units: "rhEMp (elastin and fibronectin functional module) + P3S (coiled-coil domain) and ColⅢ (recombinant type III human protein functional module) + P4S (coiled-coil domain)". Each unit is expressed independently in the E. coli chassis. This design avoids the transcriptional and translational burden on host cells from complex fusion proteins, significantly improves the expression yield of single modules, and provides technical support for industrial mass production. The extracellular matrix multimer protein (ECM-SA) obtained by this implementation method has a significantly higher L929 cell adhesion rate (100%) than rhEMp_P3S alone (74.5%), ColⅢ_P4S (71.8%), and rhECMp control (81.4%), effectively preserving high biological activity.

[0109] In a second aspect, some embodiments provide a recombinant protein composition comprising a first subunit and a second subunit of the first aspect, each separately packaged. In use, the first and second subunits of the composition are placed in a suitable environment to allow them to self-assemble into an extracellular matrix multimer protein through non-covalent interactions.

[0110] Thirdly, in some embodiments, a polynucleotide composition is provided, the polynucleotide composition comprising a first polynucleotide and a second polynucleotide; the first polynucleotide encodes a first subunit in the first aspect; and the second polynucleotide encodes a second subunit in the first aspect.

[0111] In an optional embodiment, the nucleotide sequence of the first polynucleotide is optimized according to the host cell expressing the first subunit; further optionally, the nucleotide sequence of the first polynucleotide is optimized using E. coli preferred codons.

[0112] In an optional embodiment, the nucleotide sequence of the second polynucleotide is optimized according to the host cell expressing the second subunit. Further optionally, the nucleotide sequence of the second polynucleotide is optimized using E. coli-preferred codons.

[0113] Fourthly, in some embodiments, a recombinant vector composition is provided, the recombinant vector composition comprising a first recombinant vector and a second recombinant vector; wherein the first recombinant vector carries the first polynucleotide of the second aspect, and the second recombinant vector carries the second polynucleotide of the second aspect.

[0114] In an optional embodiment, the first recombinant vector is selected from an expression vector. Further optionally, the first recombinant vector is a recombinant vector for expressing the first subunit in Escherichia coli. Further optionally, the backbone vector of the recombinant vector expressing the first subunit is derived from the pET28a(+) vector.

[0115] In an optional embodiment, the second recombinant vector is selected from the expression vector. Further optionally, the second recombinant vector is a recombinant vector for expressing the second subunit in Escherichia coli. Further optionally, the backbone vector of the recombinant vector expressing the second subunit is derived from the pET28a(+) vector.

[0116] Fifthly, in some embodiments, a recombinant cell composition is provided, the recombinant cell composition comprising a first recombinant cell and a second recombinant cell.

[0117] The first recombinant cell contains the first polynucleotide of the second aspect, or the first carrier of the third aspect, or expresses the first subunit of the first aspect. In an optional embodiment, the first recombinant cell is selected from prokaryotic cells; more preferably, the first recombinant cell is selected from *Escherichia coli*.

[0118] The second recombinant cell contains the second polynucleotide of the second aspect, or the second carrier of the third aspect, or expresses the second subunit of the first aspect. Optionally, the second recombinant cell is selected from prokaryotic cells; more preferably, the second recombinant cell is selected from *Escherichia coli*.

[0119] In a sixth aspect, some embodiments provide a method for preparing the extracellular matrix multimer protein of the first aspect, the method comprising mixing a first subunit and a second subunit, thereby causing the first subunit and the second subunit to spontaneously assemble into a multimer in a solvent.

[0120] In an optional embodiment, the freeze-dried first subunit and the freeze-dried second subunit are first mixed, and then the mixture is dissolved in a solvent, so that the first subunit and the second subunit spontaneously assemble into a polymer in the solvent.

[0121] In an optional embodiment, the mass ratio of the freeze-dried first subunit to the freeze-dried second subunit is 1:(2~3), and more preferably 1:2.3.

[0122] In an optional embodiment, the pH of the solvent is 3 to 4.

[0123] In an optional embodiment, the acid in the solvent used to provide an acidic environment includes at least one of acetic acid and citric acid. Acetic acid is preferred, as it is volatile in the subsequent lyophilization step, thus avoiding the introduction of impurities into the protein.

[0124] In an optional embodiment, the solvent is a 0.1% v / v aqueous solution of acetic acid.

[0125] In an optional embodiment, the preparation method further includes culturing the first recombinant cell and the second recombinant cell of the fifth aspect respectively, and then separating and purifying the first subunit and the second subunit.

[0126] In an optional embodiment, the preparation method further includes obtaining a first polynucleotide or a first recombinant vector for protein expression as needed, transforming the first polynucleotide or the first recombinant vector into the desired cells and expressing them.

[0127] In an optional embodiment, the preparation method further includes obtaining a second polynucleotide or a second recombinant vector for protein expression as needed, transforming the second polynucleotide or the second recombinant vector into the desired cells, and expressing them.

[0128] In an optional embodiment, the first subunit and the second subunit are purified independently using affinity chromatography or ion exchange chromatography, and further optionally, affinity chromatography purification is performed using a packing material containing nickel ions.

[0129] In a seventh aspect, some embodiments provide the use of the extracellular matrix multimer protein of the first aspect, or the recombinant protein composition of the second aspect, or the polynucleotide composition of the third aspect, or the recombinant carrier composition of the fourth aspect, or the recombinant cell composition of the fifth aspect, or the preparation method of the sixth aspect, in the preparation of formulations for use in tissue engineering, pharmaceutical or cosmetic skin care fields.

[0130] The extracellular matrix multimer protein prepared by the method of preparing the extracellular matrix multimer protein in the first aspect or the method of preparing the extracellular matrix multimer protein in the sixth aspect can be directly used as an active ingredient in formulations for use in tissue engineering, pharmaceutical or cosmetic skin care fields; the recombinant protein composition of the second aspect, or the polynucleotide composition of the third aspect, or the recombinant carrier composition of the fourth aspect, or the recombinant cell composition of the fifth aspect can be used as raw materials for producing the extracellular matrix multimer protein in the first aspect.

[0131] Eighth aspect, in some embodiments, an active additive or formulation for use in the fields of tissue engineering, pharmaceuticals, or cosmetic skincare is provided, the active additive or formulation comprising the extracellular matrix multimer protein of the first aspect, or the extracellular matrix multimer protein obtained by the preparation method of the sixth aspect.

[0132] In an optional embodiment, the active additive or formulation may further include acceptable excipients, including but not limited to one or more of carriers, diluents, buffers, protectants, stabilizers, adsorbents, matrices and excipients.

[0133] The following are some examples.

[0134] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0135] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0136] Example 1 Construction of genetically engineered Escherichia coli

[0137] 1. Construction and validation of recombinant plasmids:

[0138] (1) The design of association between parental sequence and recombinant sequence is shown in the schematic diagram. Figure 1 :

[0139] The P3S protein (SEQ ID NO.1) sequence was fused to the C-terminus of a truncated form of the extracellular matrix protein rhEMp (SEQ ID NO.2) to obtain the recombinant rhEMp_P3S protein, the sequence of which is shown in SEQ ID NO.3; the P4S protein (SEQ ID NO.4) sequence was fused to the C-terminus of recombinant type III humanized collagen ColIII (SEQ ID NO.5) to obtain the recombinant ColIII_P4S protein, the sequence of which is shown in SEQ ID NO.6.

[0140] (2) Vector construction and sequence optimization:

[0141] The commercial vector pET28a(+) was used (including the location of the functional region of pET28a(+); the sequence map of pET28a(+); the restriction enzyme sites were designed based on the relevant sequence positions of pET28a(+); and the target fragment sequence encoding rhEMp_P3S or ColⅢ_P4S protein was carried).

[0142] This embodiment uses the commercial plasmid vector pET28a(+), purchased from Guangzhou Ruibo Biotechnology Co., Ltd., and is based on... Figure 2 and Figure 3The restriction enzyme sites NdeI and EcoRI were selected for sequence design. The gene sequences encoding rhEMp_P3S or ColⅢ_P4S proteins were obtained through artificial synthesis using artificially optimized E. coli preferred codons. The full-length coding DNA fragments of the two synthesized proteins each have a restriction endonuclease site at the 5' and 3' ends, corresponding to NdeI and EcoRI, respectively. The two target fragments were inserted between the aforementioned double restriction enzyme sites on pET28a(+), which carries a His tag, resulting in expression plasmids pET28a-rhEMp_P3S and pET28a-ColⅢ_P4S encoding the recombinant proteins. The recombinant DNA fragments were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and the recombinant expression plasmids were constructed. Sequencing results showed 100% sequence identity with the theoretical sequence.

[0143] 2. Construction and Identification of Recombinant Strains

[0144] (1) Transformation of competent cells:

[0145] Escherichia coli BL21(DE3) competent cells were purchased from Beijing TransGen Biotech Co., Ltd. After thawing by placing them in an ice box for approximately 5 minutes, 1 μL of pET28a-rhEMp_P3S and pET28a-ColⅢ_P4S recombinant expression plasmids were added to two vials of E. coli BL21(DE3) competent cells, respectively. The cells were gently tapped to mix and incubated on ice for 30 minutes. The cells were then heat-shocked at 42°C for 90 seconds, quickly returned to ice to cool for 2 minutes, and 400 μL of LB liquid medium was added. The cells were then incubated at 37°C with shaking at 220 rpm for 30 minutes. Finally, the cells were evenly spread onto LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C with the plates inverted.

[0146] (2) Identification of protein expression in recombinant strains:

[0147] (a) Observe the transformation plates corresponding to BL21-rhEMp_P3S and BL21-ColⅢ_P4S strains. Several single colonies should be distributed on the plates.

[0148] (b) Pick single colonies of BL21-rhEMp_P3S and BL21-ColⅢ_P4S respectively and inoculate them into 100 mL Erlenmeyer flasks (containing 20 mL of liquid LB medium). Add 20 μL of kanamycin to the Erlenmeyer flasks and incubate at 37℃ and 220 rpm until OD600=0.5 for protein induction.

[0149] (c) Add 0.3 mM of IPTG inducer to the conical flask and incubate at 20℃ and 220 rpm for 16 h.

[0150] (d) Centrifuge 10,000 μg of fermentation broth for 2 min, discard the supernatant, and collect the cell precipitate.

[0151] (e) The collected bacterial cells were resuspended in Tris-HCl buffer (50 mM Tris-HCl, 300 mM NaCl, pH 8.0) and sonicated for 30 min (300 W power; 3 s on, 7 s off) until the solution changed from turbid to clear.

[0152] (f) After sonication, centrifuge the clarified solution at 13,000 rpm for 30 min. Remove the precipitate and retain the supernatant as the soluble protein sample.

[0153] (g) The recombinant proteins rhEMp_P3S and ColⅢ_P4S from the two supernatants in the previous step were purified by Ni-NTA affinity chromatography. The steps were as follows: the Ni-NTA affinity column was equilibrated with binding buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0), and the supernatant was loaded onto the purification column to bind the target proteins to the packing material. The target proteins were then eluted sequentially with elution buffers containing gradient concentrations of imidazole (50 mM Tris-HCl, 300 mM NaCl, 80 / 100 / 200 / 500 mM imidazole, pH 8.0).

[0154] (h) The eluent containing rhEMp_P3S and ColⅢ_P4S was poured into 10 kDa and 20 kDa ultrafiltration tubes respectively and concentrated by low-temperature, low-speed centrifugation to a volume of 0.5~2 mL.

[0155] (i) The concentrated supernatant samples containing rhEMp_P3S and ColⅢ_P4S proteins were analyzed by SDS-PAGE gel electrophoresis. The results showed specific bands at 25.8 kDa and 61.1 kDa, respectively, which were consistent with the theoretical molecular weight. Figure 4 ).

[0156] (j) rhECMp control protein, its sequence and expression identification process were performed according to CN119552268A. Protein concentration was determined using a BCA protein assay kit (Beyotime): the concentrated solutions of rhEMp_P3S and BL21-ColⅢ_P4S from step (h) and the concentrated solution of rhECMp control protein were appropriately diluted and measured and calculated according to the kit instructions. Shake-flask fermentation and target protein quantification experiments were independently repeated three times. Experimental data are presented in the form of "mean ± standard deviation (SD)". The calculated shake-flask yields of rhEMp_P3S, ColⅢ_P4S, and rhECMp control protein were 1.4 g / L, 2.6 g / L, and 0.9 g / L, respectively. Figure 5 ).

[0157] 3. Preservation of microbial strains:

[0158] Pick the single colonies that have completed the validation and transfer them to a shaker tube containing LB medium. Incubate overnight at 37°C and 220 rpm. Then take 300 μL of the bacterial culture and 300 μL of 50% glycerol, mix them thoroughly, and store the culture at -80°C.

[0159] Example 2: Preparation of ECM-SA recombinant protein element materials

[0160] 1. Shake-flask fermentation:

[0161] (a) Pick single colonies of BL21-rhEMp_P3S and BL21-ColⅢ_P4S respectively and inoculate them into 100 mL Erlenmeyer flasks (containing 20 mL LB medium) and incubate at 37℃ and 220 rpm for 16 h.

[0162] (b) Transfer the seed culture to a 2 L Erlenmeyer flask (containing 500 mL LB medium) at a ratio of 1:100, and incubate at 37°C and 220 rpm until OD600=0.5. Then add 0.3 mM IPTG and continue incubation at 20°C and 220 rpm for 16 h.

[0163] (c) After induction, collect the bacterial culture, centrifuge at 8,000 μg for 10 min, and collect the bacterial cells.

[0164] 2. Isolation and purification of rhEMp_P3S and ColⅢ_P4S proteins:

[0165] The separation and purification steps are as described in steps (e) to (h) of “(2) Identification of protein expression of recombinant strain” in Example 1. Then, ultrapure water is added to the ultrafiltration tube which is concentrated to 0.5 to 2 mL and centrifuged at low temperature and low speed. The buffer in the original system is replaced twice. The liquid in the ultrafiltration tube is taken out, frozen at -80°C overnight, and then freeze-dried into solid powder.

[0166] Example 3: In vitro formation of ECM-SA aggregates mediated by coiled helical domains

[0167] (a) The rhEMp_P3S and ColⅢ_P4S protein solids described in Example 2 are thoroughly mixed at a mass ratio of 1:2.3.

[0168] (b) Dissolve the mixed protein sample described in step (a) in a 0.1% aqueous acetic acid solution, and let it stand at room temperature for about 15 min. rhEMp_P3S and ColⅢ_P4S can spontaneously assemble into ECM-SA protein through C-terminal pairing. Figure 6 The obtained ECM-SA protein sample was analyzed by SDS-PAGE gel electrophoresis, and the results showed a main specific band at 86.9 kDa, consistent with the theoretical molecular weight. Figure 4 ).

[0169] (c) Further simulations using AlphaFold were conducted to verify the non-covalent binding between rhEMp_P3S and ColⅢ_P4S. Figure 1 ).

[0170] Example 4: ECM-SA Cell Adhesion Promotion Assay

[0171] (a) Take the individual rhEMp_P3S and ColⅢ_P4S proteins purified in Example 2 and the ECM-SA (rhEMp_P3S and ColⅢ_P4S protein complex solid) described in Example 3. Take the rhECMp control protein, the sequence of which and its preparation method are as described in CN119552268A.

[0172] (b) 0.5 mg / mL of rhEMp_P3S, ColⅢ_P4S, rhECMp and ECM-SA were added to untreated 96-well plates and then incubated at 4°C for 24 h.

[0173] (c) Add 100 μL of serum-free DMEM medium containing L929 cells (concentration 1×10⁻⁶) to each well. 5Cells were incubated at 37°C for 6–8 h (cells / mL). The culture medium was discarded, and the cells were washed three times with PBS buffer. The adherent cells in the wells were then lysed by three freeze-thaw cycles in ultrapure water. Next, Hoechst 33258 fluorescent dye was added to the cell lysis buffer at a final concentration of 5 μg / mL, and the cells were incubated in the dark for 1 h.

[0174] (d) Fluorescence intensity was measured using a microplate reader (model: Teco Infinite E Plex), with excitation wavelength set to 360 nm and emission wavelength set to 465 nm. Each sample was tested in triplicate, and experimental data are presented as mean ± standard deviation (SD). The adhesion rate of L929 cells (mouse fibroblasts) to ECM-SA was set to 100%. The adhesion rates of L929 cells to rhEMp_P3S, ColⅢ_P4S, and rhECMp were approximately 74.5%, 71.8%, and 81.4%, respectively, indicating that non-covalently assembled ECM-SA proteins based on coiled-coil domains can more effectively promote L929 cell adhesion. Figure 7 ).

[0175] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0176] 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. An extracellular matrix multimeric protein, characterized in that, comprises a first coiled coil domain and at least one extracellular matrix protein fragment; the first subunit comprises a first coiled coil domain and at least one extracellular matrix protein fragment; the second subunit comprises a second coiled coil domain and at least one extracellular matrix protein fragment; the first subunit and the second subunit are non-covalently bound by the first coiled coil domain and the second coiled coil domain.

2. The extracellular matrix multimeric protein of claim 1, wherein, the extracellular matrix protein fragments in the first subunit and the second subunit each independently comprise at least one of a collagen fragment, an elastin fragment, and a fibronectin fragment; optionally, the collagen fragment comprises a type III collagen fragment, further optionally, the type III collagen fragment comprises an amino acid sequence as set forth in SEQ ID NO. 5, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO. 5; optionally, the elastin fragment comprises an amino acid sequence as set forth in SEQ ID NO. 7, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO. 7; optionally, the fibronectin fragment comprises an amino acid sequence as set forth in SEQ ID NO. 8, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO. 8; optionally, the extracellular matrix protein fragments in the first subunit comprise the fibronectin fragment and the elastin fragment; optionally, the extracellular matrix protein fragments in the first subunit comprise an amino acid sequence as set forth in SEQ ID NO. 2, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO. 2; optionally, the extracellular matrix protein fragments in the second subunit comprise the type III collagen fragment; optionally, the first coiled coil domain is located at the C-terminus of the first subunit; optionally, the second coiled coil domain is located at the C-terminus of the second subunit; optionally, the first coiled coil domain and the second coiled coil domain comprise P3S and P4S; optionally, the first subunit and the second subunit are each independently expressed by a eukaryotic cell or a prokaryotic cell; optionally, the first subunit and the second subunit are expressed by E. coli.

3. The extracellular matrix multimeric protein according to claim 2, characterized in that, the first subunit comprises, in order from N-terminus to C-terminus, a fibronectin fragment, an elastin fragment, and a coiled coil domain P3S; and the second subunit comprises, in order from N-terminus to C-terminus, a type III collagen fragment and a coiled coil domain P4S; optionally, the first subunit comprises an amino acid sequence as set forth in SEQ ID NO. 3, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO. 3; optionally, the second subunit comprises an amino acid sequence as set forth in SEQ ID NO. 6, or a sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO. 6; Optionally, the extracellular matrix multimeric protein is a heterodimer, the amino acid sequence of the first subunit comprises the sequence as shown in SEQ ID NO. 3; the amino acid sequence of the second subunit comprises the sequence as shown in SEQ ID NO. 6; the coiled coil domain P3S is non-covalently combined with the coiled coil domain P4S; the first subunit and the second subunit are both expressed by E. coli.

4. A recombinant protein composition characterized in that, A composition comprising a first subunit as defined in any one of claims 1-3 and a second subunit as defined in any one of claims 1-3, which are independently packaged.

5. A polynucleotide composition characterized in that, A composition comprising a first polynucleotide encoding the first subunit as defined in any one of claims 1-3 and a second polynucleotide encoding the second subunit as defined in any one of claims 1-3.

6. A recombinant vector composition characterized in that, A composition comprising a first recombinant vector and a second recombinant vector; The first recombinant vector carries the first polynucleotide as defined in claim 5; The second recombinant vector carries the second polynucleotide as defined in claim 5.

7. A recombinant cell composition, characterized in that, A composition comprising a first recombinant cell and a second recombinant cell; The first recombinant cell contains the first polynucleotide as defined in claim 5, or contains the first vector as defined in claim 6, or expresses the first subunit as defined in any one of claims 1-3; The second recombinant cell contains the second polynucleotide as defined in claim 5, or contains the second vector as defined in claim 6, or expresses the second subunit as defined in any one of claims 1-3.

8. The method for producing the extracellular matrix multimeric protein according to any one of claims 1 to 3, characterized by, The method comprises mixing the first subunit and the second subunit, and allowing the first subunit and the second subunit to spontaneously assemble into a multimer in a solvent; Optionally, the method comprises mixing the lyophilized first subunit and the lyophilized second subunit, and then dissolving the mixture in the solvent; Optionally, the mass ratio of the lyophilized first subunit to the lyophilized second subunit is 1: (2-3), and further optionally 1:2.3; Optionally, the pH of the solvent is 3-4; Optionally, the acid used to provide an acidic environment in the solvent comprises at least one of acetic acid and citric acid; Optionally, the solvent is 0.1% v / v acetic acid aqueous solution; Optionally, the method further comprises separately culturing the first recombinant cell as defined in claim 7 and the second recombinant cell as defined in claim 7, and then isolating and purifying the first subunit and the second subunit.

9. Use of the extracellular matrix multimeric protein as defined in any one of claims 1-3, or the recombinant protein composition as defined in claim 4, or the polynucleotide composition as defined in claim 5, or the recombinant vector composition as defined in claim 6, or the recombinant cell composition as defined in claim 7, or the method for preparing the extracellular matrix multimeric protein as defined in claim 8, in the preparation of a preparation for the field of tissue engineering, pharmacy or cosmetic skin care.

10. An active additive or preparation for use in the field of tissue engineering, pharmacy or cosmetic skin care, characterized in that, The active additive or preparation comprises the extracellular matrix multimeric protein according to any one of claims 1 to 3, or the extracellular matrix multimeric protein obtained by the preparation method according to claim 8.

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