Recombinant I-type collagen with triple helix structure as well as preparation and application of recombinant I-type collagen

Abstract

The invention provides a recombinant I-type collagen with a triple helix structure and application thereof, and belongs to the technical field of protein engineering, the amino acid sequence of the recombinant I-type collagen is formed by repeatedly connecting n basic units in series, n is an integer greater than 1 or 1, and each basic unit comprises a Gly-Glu-Pro-Gly-Glu-Pro fragment; the invention also provides a construction method of the expression system and a preparation method of the recombinant I-type collagen. The invention also provides practical industrial application according to the characteristics of the protein. The recombinant I-type collagen provided by the invention is high in purity and has a stable triple-helix structure.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering technology, and particularly relates to recombinant type I collagen with a triple helix structure and its applications. Background Technology

[0002] The structure of natural collagen consists of three polypeptide chains forming a helical coil, which is fundamental to its biological activity. Each polypeptide chain is composed of a repeating triplet amino acid sequence of Gly-XY, where X and Y can be any amino acid. The most common triplet is proline-hydroxyproline-glycine (Gly-Pro-Hyp), accounting for approximately 10.5% of collagen triplets. Collagen is widely found in tissues such as skin, bone, and muscle, and is a major structural protein in the extracellular matrix of mammalian cells, playing various physiological functions in regulating cell proliferation, differentiation, and migration. Due to its excellent biological properties, collagen is widely used in tissue engineering, clinical medicine, the food industry, packaging materials, cosmetics, and medical aesthetics. In particular, recombinant collagen with a stable triple helix structure has great potential in the preparation of biocompatible medical materials.

[0003] Type I collagen is a heterotrimeric molecule, with each chain composed of more than 1,000 amino acids. The length of a Type I collagen molecule is approximately 300 nm, and its width is approximately 1-5 nm. Collagen trimers have high tensile strength. In most cases, it consists of two α1 chains and one α2 chain, while the α1 homotrimer exists in a minor form. Type I collagen accounts for at least 80% of the total collagen in the human body, mainly existing in the form of long fibrils in various tissues and vascular systems such as skin, tendons, ligaments, bones, lungs, cornea, and organ capsules. Summary of the Invention

[0004] In view of this, the first objective of the present invention is to provide a recombinant type I collagen with a triple helix structure. The recombinant collagen provided by the present invention has an advanced structure, high purity, and good thermal stability.

[0005] Specifically, the recombinant type I collagen of the present invention is obtained by connecting and repeatedly tandemly linking multiple truncated regions. For example, the recombinant collagen of the present invention is obtained by truncating human type I collagen between 1-100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, or 1-3 amino acids.

[0006] The present invention discloses a recombinant type I collagen whose amino acid sequence is formed by repeated tandem of n basic units, where n is an integer greater than 1, and the basic unit contains a Gly-Glu-Pro-Gly-Glu-Pro fragment.

[0007] Preferably, the amino acid sequence of the basic unit is as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

[0008] It should be understood that this invention essentially protects the amino acid sequence of recombinant type I collagen. Therefore, the mass production of proteins containing basic unit sequences in the form of single-chain structures still falls within the protection scope of this invention.

[0009] The value of n is preferably selected from any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and more preferably from any one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

[0010] The n basic units are repeatedly connected in series, either by connecting the basic units end to end or by connecting the basic units in series by a linker. The linker preferably contains glycine and / or serine, and preferably contains 1-5 amino acids.

[0011] Furthermore, the amino acid sequence formed by the tandem connection of the basic units described in this invention may also have multiple amino acids spliced ​​at its head (N-terminus) and tail (C-terminus), preferably 1-2 amino acids.

[0012] More preferably, the basic units with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 are tandemly repeated head and tail to form recombinant collagen with amino acid sequences as shown in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, and are named COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160 and COL161, respectively.

[0013] In this invention, the recombinant type I collagen can be used as a domain to connect with domains from other sources to form a fusion protein, thereby constructing and expressing a novel target protein with multiple functions; it can also be linked with tags that help with protein expression, purification or detection.

[0014] In this invention, the introduction of N-terminal amino acids into the protein can be carried out in accordance with the method described in CN117511978A, which enhances the stability of the RNA secondary structure, slows down the protein translation rate, and allows the peptide chain to better form the target protein, thereby increasing the yield of exogenous protein.

[0015] In this invention, the amino acid introduced at the C-terminus of the protein can be an amino acid derived from human type I collagen.

[0016] This invention provides a gene encoding the recombinant type I collagen described above.

[0017] The encoding includes proteins produced by transcription of DNA molecules to form an RNA product, followed by translation; or proteins produced by transcription of DNA molecules to provide an RNA product, processing to provide a processed RNA product, followed by translation.

[0018] The present invention provides a recombinant vector expressing the recombinant type I collagen, comprising the aforementioned gene.

[0019] Vectors include any nucleic acid molecule derived from any source and capable of genome integration or autonomous replication (e.g., plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), comprising nucleic acid molecules operatively linked with one or more nucleic acid molecules. Vectors may include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome. Preferably, the vectors include, but are not limited to, pPICZαA and pPIC9k.

[0020] In this invention, the vector can be cloned with the recombinant human type I collagen gene alone, or it can be cloned with the proline hydroxylase gene and / or the lysine hydroxylase gene and the recombinant human type I collagen gene on the same vector.

[0021] The present invention provides recombinant cells expressing the recombinant type I collagen, wherein the recombinant cells are transfected with the recombinant vector.

[0022] The recombinant cells include, but are not limited to, Escherichia coli, yeast, mammalian cells, and plant cells.

[0023] The *E. coli* strain is BL21(DE3). The recombinant type I collagen encoding gene can be expressed alone or co-expressed in *E. coli* with the viral lysyl hydroxylase L230 gene, proline hydroxylase L593 gene, and / or *Bacillus anthracis* P4H gene. The yeast strain is *Pichia pastoris* X-33 or GS115. The mammalian cells can be Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK293) cells, or the fibrosarcoma cell line HT1080.

[0024] Methods for obtaining transduced cells expressing protein molecules by introducing a vector into other host cells are known. Similar methods can be used in this invention.

[0025] A second objective of the present invention is to provide a method for expressing the recombinant type I collagen.

[0026] In a preferred embodiment, the recombinant type I collagen is expressed in Pichia pastoris GS115, comprising the following steps:

[0027] 1) The gene was ligated into the vector pPICZαA to obtain a recombinant vector;

[0028] 2) The recombinant vector was transfected into Pichia pastoris GS115 to obtain recombinant cells;

[0029] 3) After culturing the recombinant cells, centrifuge and collect the supernatant;

[0030] 4) The recombinant type I collagen was obtained by separating it from the supernatant.

[0031] A third objective of this invention is to provide the application of the recombinant type I collagen and the recombinant type I collagen prepared by the aforementioned method in the preparation of medical devices or daily chemical products.

[0032] Triple helix structure

[0033] Collagen, as a major component of the natural extracellular matrix, provides good support for cells with its unique triple helix structure and collagen fibers, creating a suitable microenvironment for cell proliferation and migration.

[0034] Typical collagen is a superhelical molecular structure formed by three intertwined α-peptide chains in a left-handed helical conformation. The middle part is a continuous triple helix, while the peptide chains at both ends of the molecule exhibit a random coil conformation, which plays a crucial role. Other techniques, such as NMR, have also advanced the study of sequence dependence of triple helix conformation characteristics. The triple helix structure has now been confirmed to be important for many specific biological interactions and as a structural element.

[0035] However, current animal-derived collagen often suffers from varying degrees of damage to its natural cross-linked structure due to complex extraction methods or the introduction of excessive chemical reagents. Furthermore, many commercially available recombinant collagens lack a triple helix structure or sufficient mechanical strength, making them easily hydrolyzed by proteases in the body after implantation. Therefore, this invention aims to optimize the structure and performance of recombinant collagen by introducing more stable components, such as improved thermal stability, to ensure that the collagen raw material possesses a certain level of mechanical strength and maintains a degree of "inertness" against proteases.

[0036] This invention characterizes the structure of collagen using circular dichroism (CD), a commonly used spectroscopic method in the field. CD is used to determine the structure of compounds with chiral structures that produce differential absorption between left and right rotations, and is mainly used to determine the asymmetry of molecular structures. Most biological macromolecules contain chiral groups and structures; therefore, CD is often used to measure and observe changes in the structure and conformation of biological macromolecules. The CD characteristic of the triple helix structure of collagen generally shows a positive absorption peak near 221 nm and a negative absorption peak near 195 nm (industry standard YY / T1849—2022). The positions of the absorption peaks shift with changes in the amino acid sequence and length. The thermal stability of collagen includes the thermal shrinkage temperature (Ts) and the thermal denaturation temperature (Td) of collagen fibers. The thermal shrinkage temperature of collagen fibers refers to the temperature at which collagen fibers shrink axially to about 5% of their original length when heated. The thermal denaturation temperature of collagen refers to the temperature at which the triple helix structure of collagen unwinds and forms individual single chains when heated in a medium to a certain temperature, reaching 50% unwinding. Therefore, CD spectroscopy can be used to study the helical structure of collagen and its thermal denaturation process.

[0037] Collagen degradation characteristics

[0038] Trypsin is a serine protease whose specific recognition site is the carboxyl-terminal peptide bond of lysine (K) and arginine (R). In the in vitro degradation of recombinant collagen, trypsin precisely binds to the side chains of K / R residues via a catalytic triplet (histidine, aspartic acid, serine) at its active site, hydrolyzing the peptide bond formed at the carboxyl terminus. Trypsin first disrupts the tertiary structure of collagen, depolymerizing its triple helix conformation, and then cleaves the polypeptide chain into smaller fragments containing K / R ends. The sensitivity of recombinant collagen to trypsin varies significantly depending on the design strategy (e.g., amino acid sequence, cross-linking modification, molecular weight).

[0039] Collagenase is a protease that specifically degrades the triple-helix structure of natural collagen. Its unique characteristic lies in its ability to recognize and cleave the characteristic sequence Pro-X-Gly-Pro (PXGP, where X represents a neutral amino acid) in collagen, a highly ordered structure that ordinary proteases cannot degrade. Collagenase precisely binds to the PXGP sequence in the triple-helix region of collagen through its catalytic domain. This sequence appears frequently in collagen but is rare in other proteins. Collagenase hydrolyzes the peptide bonds between X (such as neutral amino acids like alanine and leucine) and glycine (Gly), breaking the long collagen chain into smaller fragments. Some domains of collagenase (such as the collagen-binding domain CBD) can locally disrupt the hydrogen bond network of the triple helix, loosening the tightly wound peptide chains and exposing more cleavage sites. The degradation rate and extent of recombinant collagen are significantly influenced by its structural design, modification methods, and physicochemical properties. For example, recombinant collagen lacking hydroxylase modification (as in some E. coli expression systems) or with low thermal stability exhibits increased helical looseness and a faster degradation rate. Through data analysis, sequence design, and kinetic simulation, the inventors discovered that anti-collagenase degradation requires both the absence of characteristic sequences and a tight triple helix structure. This gives the recombinant collagen of this invention the following characteristics: (1) Long-lasting efficacy: It stays in the skin or wound for a longer time, enabling it to exert its biological activity of promoting cell adhesion, proliferation, and differentiation more continuously, providing long-lasting repair, moisturizing, and support effects; (2) High bioavailability: It has more time to bind to receptors on the cell surface, stimulating the cell's own collagen production signals, thereby exerting its biological function more efficiently; (3) Lower immunogenicity: As mentioned above, the removal of animal-derived telopeptides and viral risks makes it highly compatible with the human body, safer, and suitable for long-term use by a wider range of people.

[0040] Skin homeostasis-related factors

[0041] Aquaporin 3 (AQP3) is a major water-glycerol transporter expressed in epidermal keratinocytes, responsible for maintaining the osmotic pressure balance and moisturizing function of the skin barrier. Its expression level is directly related to skin hydration, elasticity, and wound repair efficiency. Studies have shown (Yuying Zhu et al., 2024) that collagen can upregulate AQP3 expression by activating the integrin-EGFR signaling axis, thereby enhancing cell membrane permeability to water and glycerol and improving dry skin. Laminin 5 is a core component of the basement membrane, encoded by the LAMA3, LAMB3, and LAMC2 genes. It maintains skin mechanical stability by forming anchoring fibers that connect the epidermis and dermis. In the embodiments of this invention, recombinant collagen exhibits activity that can upregulate the expression of skin homeostasis-related factors, which is beneficial for resisting skin aging and promoting damage repair.

[0042] Inflammatory response and mechanism

[0043] The mechanism by which H2O2 induces oxidative damage and inflammatory responses is mainly due to the cytotoxic effects of H2O2. As a core carrier of reactive oxygen species (ROS), the hydroxyl radicals (-OH) generated by the decomposition of H2O2 attack the phospholipid bilayer of the cell membrane, generating malondialdehyde (MDA), which disrupts membrane integrity and releases arachidonic acid, activating the COX-2 / PGE2 pathway and promoting prostaglandin E2 synthesis. H2O2 inhibits the activity of mitochondrial complex I / III, leading to increased electron leakage in the electron transport chain, further amplifying ROS generation and forming a vicious cycle. The collapse of the mitochondrial membrane potential (ΔΨm) releases cytochrome C, activating...

[0044] The Caspase-9 / 3 apoptosis pathway is activated, simultaneously inducing mitochondrial DNA oxidative damage and releasing mtDNA fragments as damage-associated molecular patterns (DAMPs), activating the TLR9 / NF-κB inflammatory pathway. H2O2 inhibits the ubiquitination and degradation of IKKβ, promoting IκBα phosphorylation and dissociation of the NF-κB dimer (p50 / p65). Free p65 enters the nucleus and binds to the κB site (GGGRNNYYCC) in the promoter region of inflammatory cytokine genes, driving the transcription of TNF-α, IL-6, and IL-1β, thus activating the NF-κB pathway. H2O2-induced K... + Efflux and mitochondrial ROS burst promote the binding of NLRP3 to ASC protein, activate Caspase-1, and then cleave pro-IL-1β into its active form, amplifying inflammatory signals.

[0045] The criteria for selecting inflammatory cytokine targets are shown in the table below:

[0046] Inflammatory factors Core biological functions Pathological role location IL-1α Tissue damage "alarm hormones" and innate immune activation Early stage of local inflammation amplifier IL-8 Neutrophil chemotaxis and activation core factors Drivers of acute inflammatory cell infiltration TNF-α Inflammation initiation hub, apoptosis / necrosis induction Systemic cytokine storm core mediator COX-2 terminal enzymes in the synthesis of prostaglandin inflammatory mediators Direct effector molecules of pain / fever / vascular permeability

[0047] In vivo, collagen may activate the downstream PI3K / Akt / Nrf2 pathway by specifically binding to HaCaT cell surface receptors, such as integrin α2β1, promoting Nrf2 nuclear translocation and binding to antioxidant response elements (AREs), upregulating the expression of enzymes such as HO-1, SOD, and CAT, and directly neutralizing H2O2. It may also utilize DDR1 (disc-domain receptor 1), where collagen binding induces receptor dimerization, phosphorylates the intracellular domain, recruits Shc adaptor proteins, inhibits the ASK1 / JNK pathway, blocks JNK's ability to phosphorylate c-Jun, and reduces AP-1-driven transcription of inflammatory genes. It can also clear H2O2-damaged mitochondria by restoring PINK1 / Parkin-mediated mitophagy, blocking DAMPs release. Furthermore, it can inhibit H2O2-induced PARP1 overactivation, reduce PINK1 parylation modification, and maintain its ability to accumulate on the mitochondrial outer membrane. Degradation products of recombinant type I collagen (such as Gly-Pro-Hyp tripeptide) bind to the SIRT1 catalytic domain, enhancing its deacetylation activity, reducing the H3K9ac modification level in the promoter region of inflammatory genes or activating DNMT3B, increasing methylation modification on CpG islands 1.5kb upstream of the TSS of the TNF-α gene, and hindering p65 binding.

[0048] Subcutaneous injection filler

[0049] Subcutaneous filler injection is a technique that involves injecting filler material into appropriate locations and quantities via percutaneous injection to achieve cosmetic results. Ideal soft tissue filler materials should possess good safety, biocompatibility, and effectiveness. Currently, facial minimally invasive filler materials are primarily non-autologous tissue injectables, such as single-component fillers like hyaluronic acid (HA) and collagen. Collagen is widely used in aesthetic medicine to repair skin defects and subcutaneous diseases. Local injection of collagen into the face can achieve effects such as facial contour correction, wrinkle reduction, and scar repair. Recombinant collagen fillers, due to their resistance to enzymatic degradation, have a significantly extended retention time in the body (theoretically up to 12 months or even longer), providing a more lasting supporting and filling effect, reducing the number of injections, and lowering the risk of infection.

[0050] Medical repair materials

[0051] Collagen materials possess a natural porous network structure, which allows them to rapidly absorb blood upon contact with the wound, promoting platelet aggregation. Simultaneously, they absorb tissue exudate, creating a favorable wound microenvironment and accelerating wound healing. Currently, there are numerous collagen-based soft tissue repair products available clinically, including collagen dressings, dura mater patches, artificial corneas, oral patches, hernia patches, and breast augmentation patches. Their applications include, but are not limited to, wound healing, skin repair, dermatitis, eczema, hemorrhoids, oral mucositis, oral ulcers, scars, and allergic rhinitis.

[0052] Based on this, the present invention provides a method for promoting wound repair. The method includes identifying tissue requiring repair, such as collagenous tissue (e.g., cornea, skin, bone), connective tissue (e.g., cartilage, ligaments, tendons, or basement membrane); and contacting the tissue with an agent that increases type I collagen. Wounds can be caused by trauma (e.g., connective tissue or muscle tears), incisions, or surgeries (e.g., transplants), such as heart transplants, lens transplants, joint replacements, hair follicle replacements, and skin grafts. In one scenario, the wound is used to repair wounds present in the eye, such as surgical incisions, corneal transplants, LASIK flap reconnection, cataract surgery, laser surgery, corneal transplants, penetrating keratoplasty, posterior lamellar keratoplasty, refractive surgery, corneal remodeling, or treatment of corneal lacerations. For example, the wound may be present in heart tissue, a heart valve, or further, as a result of heart valve replacement or transplantation. For example, the wound may be present in cartilage, tendons, or ligaments. For example, the wound may be present in epithelial tissue, such as in the dermis.

[0053] In one specific case, collagen sponge artificial dura mater was applied to patients with post-traumatic epilepsy. It was found that the collagen sponge artificial dura mater could effectively prevent cerebrospinal fluid leakage, had a good hemostatic effect, and did not increase the incidence of epilepsy. No infection or rejection reaction occurred in the patients after the operation, indicating that it has good tissue compatibility and can be used for intracranial tumors (including gliomas, meningiomas, pituitary adenomas, schwannomas and other congenital tumors, metastatic cancers, etc., intracranial aneurysms and vascular malformations, spinal tumors, and other lesions, germ cell tumors, epileptic foci, colloid cysts, inflammatory granulomas, etc. (Qin Guoqiang et al., 2014).

[0054] In another specific example, collagen, as an absorbable suture, is used for wound suturing. It can provide sufficient nutrition for wound healing and has been clinically tested and scientifically proven to have excellent properties such as complete absorption without scarring, ease of use, good biocompatibility, no tissue rejection reaction, and appropriate absorption time.

[0055] Medical transplant materials

[0056] Tissue engineering applies engineering and life science principles to the research of biological substitutes. It utilizes seed cells, scaffolds, and growth factors to construct tissues and organs similar to those of the human body, which are then transplanted back into the host to regenerate the missing or damaged tissues and organs, forming new tissues that closely resemble normal tissues. This restores the damaged shape or partial function, achieving the goal of regenerating damaged tissues and organs. The transplanted materials can be organs or parts thereof, such as artificial eyes or lenses, artificial hearts or heart valves, artificial hair follicles, artificial muscles; they can be artificial connective tissues, such as artificial cartilage, tendons, ligaments, or basement membrane tissue; artificial bone tissue; an artificial tooth; or a prosthesis. In a preferred embodiment, the transplanted tissue is eye tissue, such as a lens; heart tissue, such as a heart valve; cartilage tissue, such as tendons; muscle tissue; hair follicles; or epithelial tissue, such as skin grafts.

[0057] Type I fibrillary collagen is the main collagen in the heart muscle (Mahboubeh Eghbali, 1990). Type I collagen or functional fragments thereof may help treat diseases of heart valves or other areas of the heart, or be used to manufacture, treat, or maintain transplantable bioequivalents of parts of the heart. Type I collagen may help improve the success rate of such transplants, or extend the lifespan of the heart and its valves, or improve the success rate of cardiac surgery.

[0058] Because type I collagen is expressed at the muscle-connective tissue boundary of the eye, type I collagen or functional fragments thereof may help treat eye diseases or be used to create, treat, or maintain transplantable bioequivalents of parts of the eye. Type I collagen can be used for such transplants, such as lens replacement surgery, or to extend the lifespan of the lens and its tissues, or to increase the success rate of ophthalmic surgeries, such as treating surgically induced endophthalmitis.

[0059] Mature dentin contains approximately 70% hydroxyapatite, 18% collagen, 2% non-collagenous proteins, and 10% water. COLⅠ (Collagen-Oxygen Composite) is the scaffold structure of the dentin matrix, combining collagen with hydroxyapatite to form a hydroxyapatite-mineralized collagen complex, which can be used as a osteoconductive coating and scaffold. For example, in dental restorations, collagen fills periodontal bone defects, accelerating periodontal bone hyperplasia and gingival regeneration (Thunwa Binlateh, 2022). Furthermore, collagen is the most abundant matrix protein in bone tissue.

[0060] Daily chemical products

[0061] Compositions containing recombinant collagen can be applied topically to the skin to reduce skin damage or promote the repair of damaged skin. For example, the compositions can protect the skin from UV damage, protect skin cells from the effects of exposure to oxidation, promote the repair of damaged skin by increasing cell vitality and / or increasing procollagen synthesis when applied to the skin, and / or promote the vitality of skin cells.

[0062] Based on this, the present invention can select a composition comprising at least one of the aforementioned proteins in a cosmetically or pharmaceutically effective amount, and at least one excipient or cosmetically or pharmaceutically acceptable adjuvant, depending on the actual application. The dosage form of the composition includes, but is not limited to, creams, lotions, aqueous solutions, gels, oils, powders, muds, patches, films, or lyophilized forms.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] 1) The recombinant type I collagen of the present invention is based on the original amino acid sequence of human type I collagen. Its molecular weight is significantly smaller than that of natural full-length collagen (300kDa). It has optimized functional active sites to fully exert biological functional activity.

[0065] 2) The recombinant type I collagen of this invention has a triple helix structure consistent with natural collagen, and its thermal stability is higher than that of human body temperature and commercially available products, which is beneficial to industrialization and downstream applications.

[0066] 3) The recombinant type I collagen of this invention has good biocompatibility and resistance to in vitro degradation, making it an excellent material for medical fillers. Attached Figure Description

[0067] Figure 1 This is a fermentation supernatant diagram of the recombinant type I collagen COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, and COL161 of this invention.

[0068] Figure 2 This is an SDS-PAGE image of the purified recombinant type I collagen COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, and COL161 of this invention.

[0069] Figure 3 The circular dichroism chromatograms of the recombinant type I collagen COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, and COL161 of this invention at room temperature are shown.

[0070] Figure 4The circular dichroism chromatograms of the recombinant type I collagen COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, and COL161 of this invention at different temperatures are shown.

[0071] Figure 5 The results of in vitro enzymatic hydrolysis experiments of recombinant type I collagen COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, and COL161 of this invention are shown.

[0072] Figure 6 This is a diagram showing the cell adhesion activity results of the recombinant type I collagen COL152 of this invention.

[0073] Figure 7 This is a graph showing the cell proliferation activity results of the recombinant type I collagen COL152 of this invention.

[0074] Figure 8 This diagram shows the regulatory effects of the recombinant type I collagen COL152 of this invention on the AQP3 and Laminin5 genes in human fibroblasts.

[0075] Figure 9 This diagram shows the regulatory effects of recombinant type I collagen COL152 of this invention on collagen and vascular endothelial factor genes in human fibroblasts and human keratinocytes.

[0076] Figure 10 This diagram shows the regulatory effects of recombinant type I collagen COL152 on matrix metalloproteinase genes in human fibroblasts and mouse fibroblasts.

[0077] Figure 11 This diagram illustrates the regulatory effects of recombinant type I collagen COL152 of this invention on inflammatory factor genes in human keratinocytes after oxidative damage. Detailed Implementation

[0078] In this invention, the gene can be synthesized by a biotechnology company. This invention does not impose any particular limitation on the preparation method of the recombinant vector and recombinant cells; conventional methods for preparing recombinant vectors and recombinant cells in the art can be used.

[0079] The present invention does not specifically limit the separation and purification method, and conventional electroporation, yeast culture, and protein separation and purification methods in the art can be used; preferred technical solutions are described in the embodiments.

[0080] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0081] Example 1: Expression, isolation, and purification of recombinant type I collagen of the present invention

[0082] 1. Sequence synthesis

[0083] GenScript Biotech Inc. was commissioned to synthesize gene fragments of the recombinant type I collagen (COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, COL161) of this invention, and construct them into the pPICZalphaA Pichia pastoris expression vector.

[0084] 2. Construction of Pichia pastoris expression system

[0085] 2.1 Electric Rotor

[0086] Plasmid pPICZalphaA was amplified by PCR and linearized at the SacI site, followed by PCR fragment recovery (ThermoGeneJET PCR Purification Kit). Competent GS115 cells were thawed on ice, and 5-10 μg of the linearized plasmid was added. The plasmid was then electroporated into competent cells. Immediately after electroporation, 1 mL of pre-chilled sorbitol was added. The cells were centrifuged at 5000g, resuspended, and plated onto YPDSZ agar plates (tryptone 20 g / L, yeast extract 10 g / L, D-sorbitol 182.1 g / L, 20% glucose, 100 μg / mL bleomycin). The plates were incubated at 30°C and 230 rpm for 48 h.

[0087] 2.2 Remove the plate containing colonies, pick out single colonies, and copy them onto YPD solid medium containing bleomycin for screening.

[0088] 2.3 Remove the plates containing colonies, pick single colonies, and inoculate them into 50ml LYPD liquid medium (containing bleomycin) for overnight seed culture at 30℃ and 230rpm. Inoculate the overnight seed culture at a 1:50 ratio into 1L BMGY medium (tryptone 20g / L, yeast extract 10g / L, potassium dihydrogen phosphate (KH2PO4) 11.73g / L, dipotassium hydrogen phosphate (K2HPO4) 2.4g / L, glycerol 10g / L, YNB 134g / L, and 1×biotin) for scale-up culture. Centrifuge the BMGY culture at 3000rpm for 5min, discard the supernatant, resuspend in sterile water, centrifuge again at 3000rpm for 5min, discard the supernatant, resuspend in 1L BMMY medium (without methanol), and add 1% methanol for induction. After induction for 72 hours, the supernatant was collected by centrifugation and purified. The SDS-PAGE results of the fermentation supernatant are as follows: Figure 1 As shown.

[0089] 2.4 Ion exchange purification

[0090] The target protein was dialyzed into buffer A (20 mM PB, pH 4.0). The protein sample was filtered through a 0.22 μm pore size and 13 mm diameter filter membrane, followed by cation exchange using an AKTApure column (5 mL SP column). Linear elution with buffer B (20 mM PB and 1 M NaCl, pH 7.4) yielded a highly pure target protein. SDS-PAGE electrophoresis was used to verify the protein's molecular weight and purity. Protein loading buffer (with added DTT) was added to the protein sample, mixed well, and placed in a metal bath. The mixture was heated at 95°C for 10 min, centrifuged at 1500 rpm for 1 min, and the supernatant was analyzed by SDS-PAGE. The results are shown below. Figure 2 As shown. Protein concentration was determined by BCA method to estimate protein yield. Based on protein purity, concentration, liquid replacement, and lyophilization were performed for preservation.

[0091] Example 2: Circular dichroism (CD) detection of structural features of recombinant type I collagen

[0092] The recombinant type I collagen lyophilized powder prepared in Example 1 was dissolved in 20 mM PB buffer (pH 7.4) to a concentration of 0.5 mg / mL. The sample was further diluted to a concentration of 0.01 mg / mL and a volume of 3 mL. The sample was transferred to a 10 mm × 10 mm sample cell of a circular dichroism chromatograph. The scanning wavelength range was set to 190 nm–260 nm, the scanning speed to 100 nm / min, and the scanning temperature to room temperature. The CD spectra were the average of three scans. Collagen has three left-handed α-helical peptide chains, which are wound in parallel, right-handed helical configurations to form a "rope-like" triple helix structure. This structure exhibits a characteristic absorption spectrum in the far ultraviolet region (185–250 nm), with a negative peak around 195 nm and a positive peak around 221 nm. The results are as follows: Figure 3 As shown, the recombinant type I collagen of the present invention has a maximum characteristic positive peak at 221 nm and a negative peak at 195 nm. Based on the known circular dichroism chromatographic characteristics of the triple helix structure of collagen, the recombinant type I collagen samples COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, and COL161 of the present invention all have a triple helix structure.

[0093] The Circular Dichroism (RPN) value is calculated by measuring the CD value (circular dichroism) of a collagen solution at a specific wavelength. Specifically, the RPN value is the ratio of the absolute values ​​of the positive peak intensity to the negative peak intensity, i.e., RPN = positive peak intensity / |negative peak intensity|. Changes in the RPN value can reflect changes in collagen structure to some extent. When the triple helix structure of collagen is significantly disrupted, the RPN value drops to 0 or even negative. For example, the RPN value of fully denatured collagen or gelatin is 0 or even negative. During heating, the triple helix structure of collagen is affected, leading to changes in the RPN value. The main reason is that increased temperature affects the hydrogen bonds within the collagen molecule, thus affecting its conformation. The RPN value can be used to assess the structural integrity and degree of denaturation of collagen. By monitoring changes in the RPN value, the structural stability of collagen under different conditions (such as heating, irradiation, etc.) can be understood.

[0094] The RPN values ​​of the recombinant type I collagen samples COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, and COL161 of the present invention are shown in Table 1.

[0095] Table 1

[0096] Recombinant Type I Collagen Positive peak intensity negative peak intensity RPN COL134 2.234 31.824 0.070 COL143 1.179 30.773 0.026 COL152 11.413 149.813 0.076 COL153 8.293 107.200 0.077 COL155 0.745 73.597 0.010 COL159 3.938 62.684 0.063 COL160 4.920 75.737 0.065 COL161 3.941 57.401 0.069

[0097] Example 3: Real-time thermal stability of recombinant type I collagen determined by circular dichroism (CD) chromatography.

[0098] The collagen sample was diluted according to the method described in Example 2, and the thermal stability of the protein was tested by real-time temperature increase at a rate of 1℃ / min. The CD spectra were the average of three scans, and the data were analyzed and calculated using software. The results are as follows: Figure 4 As shown, the disappearance of the characteristic positive peak at 221nm is used as the criterion to indicate that the triple helix structure formed by the recombinant collagen of the present invention is relatively stable. Among them, the thermal stability of COL134, COL152, COL159 and COL161 is greater than 37℃, which is beneficial for use as implantation, filling and repair materials.

[0099] Example 4: In vitro anti-degradation experiment to detect the stability of recombinant type I collagen

[0100] Recombinant type I collagen lyophilized powders (COL134, COL143, COL152, COL153, ​​COL155, COL159, COL160, COL161) prepared in Example 1 and commercially available bovine type I collagen (animal-derived extract) were dissolved in PBS buffer to prepare solutions with a concentration of 1.0 mg / mL and allowed to equilibrate to room temperature. Trypsin (purchased from Promega) and collagenase (purchased from Beyotime) were added to each collagen solution to make the mass ratio of trypsin to substrate 1:500. The mixtures were incubated in a 37°C constant temperature water bath, and samples were taken at time points of 0 min, 10 min, 20 min, 30 min, 1 h, 2 h, and 4 h. The collagenase-substrate mass ratio was 1:20. The mixture was incubated in a 37℃ water bath, and samples were taken at 0 min, 30 min, 2 h, 4 h, 24 h, 48 h, and 72 h. 20 μL of the reaction solution was added to protein loading buffer containing DTT, and the mixture was immediately vortexed. The sample was then heated in a metal bath at 95℃ for 10 min, followed by centrifugation at 1500 rpm for 1 min. The supernatant was collected for later use. 10 μL of the supernatant was subjected to SDS-PAGE electrophoresis (conditions: constant voltage 160 V, electrophoresis time 40 min). Coomassie brilliant blue staining was performed to observe the degradation of the target protein. Results ( Figure 5 This indicates that the recombinant type I collagen prepared by this invention has a certain resistance to enzymatic hydrolysis compared to animal-derived products, making it suitable for biomedical applications requiring long-term stability (such as tissue engineering scaffolds or sustained-release formulations). Among these, COL143, COL155, COL159, and COL160 can better prolong collagenase hydrolysis, facilitating the preparation and application of suture and implant materials; COL134, COL160, and COL161 have a wider range of applications, especially in applications resistant to common proteases in the environment.

[0101] Based on the thermal stability results identified by circular dichroism spectroscopy, the thermal stability of COL134, COL152, COL159, and COL161 is all greater than 37℃. The triple helix structure is more tightly wound, which gives them a certain degree of resistance to enzymatic degradation. Therefore, the stability of the triple helix structure is the main reason for its resistance to trypsin degradation.

[0102] Example 5: In vitro cell function experiment to detect the adhesion activity of recombinant type I collagen

[0103] Cell adhesion is a core prerequisite for tissue integrity and also serves as a regulatory mechanism for the expression of functional proteins in cells. It mainly includes two aspects: intercellular adhesion and cell adhesion to the external matrix. The specific steps are as follows:

[0104] 1. Coating Culture Plates: Take 96-well cell culture plates and coat them according to the following groups (100 μL / well). The experimental groups were prepared by diluting recombinant type I collagen COL152 with phosphate-buffered saline (PBS, pH 7.4) to final concentrations of 0 mg / mL, 0.41 mg / mL, 2.03 mg / mL, 4.07 mg / mL, 8.13 mg / mL, 16.27 mg / mL, 24.40 mg / mL, 32.53 mg / mL, and 40.66 mg / mL. The control groups were prepared by using PBS buffer and bovine serum albumin (BSA, 5% w / v solution) as negative controls. Commercially available bovine type I collagen was used as a competitor product and was diluted with PBS to a final concentration of 5 μg / mL. The plates were incubated at 4°C for 16-18 hours (overnight).

[0105] 2. Blocking non-specific binding sites: Discard the coating solution, add 200 μL of DMEM medium containing 1% BSA to each well, and block at 37°C for 1 hour.

[0106] 3. Cell seeding and culture: Prepare a suspension of 3T3 mouse fibroblasts (density: 5×10^4 cells / mL), seed 100μL per well (i.e., 5000 cells / well), and incubate at 37℃ in a 5% CO2 incubator for 24 hours.

[0107] 4. Culture for a period of time: Culture for 24 hours so that the cells can adhere to the collagen.

[0108] 5. Wash away unattached cells: Aspirate the culture medium and gently wash three times (200 μL each time) with PBS buffer pre-warmed to 37°C to remove unattached cells.

[0109] 6. Quantitative detection of cell adhesion: Add 110 μL of mixed solution (containing 100 μL DMEM basal medium + 10 μL LCK-8 reagent) to each well, incubate at 37°C in the dark for 2 hours, and detect the absorbance using a multi-mode microplate reader. Main wavelength: 450 nm, reference wavelength: 650 nm (dual wavelength correction to eliminate background interference).

[0110] 7. Data Analysis and Calculation: Calculate the relative cell adhesion rate based on the absorbance value.

[0111] Relative cell adhesion rate (%) = [(A exp -A blank ) / (A ctrl -A blank )]×100%

[0112] In the formula:

[0113] A exp : Absorbance of the experimental group (COL152 coated wells);

[0114] A ctrl : Absorbance of control group (competitor product or BSA-coated well);

[0115] A blank Absorbance of cell-free blank wells (containing only CCK-8 mixture):

[0116] The results are as follows Figure 6 As shown, the recombinant type I collagen COL152 of the present invention exhibits certain adhesion activity at high concentrations (≥4.07 mg / mL).

[0117] Example 6: In vitro cell experiments to evaluate the safety of recombinant type I collagen

[0118] Mouse L929 cells (Chinese Academy of Sciences Cell Bank) were cultured at 37°C and 5% CO2 in DMEM medium (Pronosai) containing 10% fetal bovine serum (FBS, Sijiqing) until the logarithmic growth phase (cell density reached 80–90% confluence). The original medium was discarded, and the cells were washed twice with PBS (pH 7.4). 1 mL of 0.25% trypsin-EDTA solution (Gibco) was added, and the cells were digested at 37°C for 2 minutes (microscopic confirmation of cell retraction and detachment was obtained). Digestion was terminated by adding complete medium containing 10% FBS, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in complete medium. The cell suspension was diluted to a density of 5 × 10^4 cells / mL and seeded into 96-well plates. 100 μL (5000 cells / well) was added to each well. 200 μL of PBS buffer was added to the outer 36 wells, and only the central 60 wells were used for experimental samples to minimize evaporation error. After inoculation, cells were pre-cultured for 24 hours in a Thermo incubator at 37℃, 5% CO2, and ≥90% humidity to confirm cell adhesion >95%. The original culture medium was discarded, and recombinant type I collagen COL152 diluted with fresh culture medium to final concentrations of 0 mg / mL, 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, and 10.0 mg / mL (200 μL per well) were added sequentially. Incubation continued for 48 hours (time error ±0.5 hours). CCK8 reagent (Beyotime) was diluted to 10% (v / v) with complete culture medium and used immediately. Add 10 μL of CCK8 stock solution (final concentration 10%) directly to each well, or discard the culture medium in the well and add 110 μL of fresh culture medium containing 10% CCK8 (to avoid interference from the original culture medium components). Continue incubation for 1-4 hours (preferably 2 hours) until the negative control group shows an orange-yellow color (OD 450-490 nm ≥ 0.8). Perform dual-wavelength detection using a microplate reader, detecting at wavelengths of 450 nm (main peak) and 490 nm (auxiliary peak, excluding impurity interference), with a reference wavelength of 650 nm (subtracting background scattering). Repeat reading each well 3 times, take the average value, and record the OD value (accuracy ± 0.001).

[0119] Calculation formula:

[0120] Cell proliferation rate (%) = [(OD)] 实验组 -OD 空白组 ) / (OD 阴性对照组 -OD 空白组 )]×100%

[0121] In the formula:

[0122] Control group: Add 200 μL of PBS to each well (to avoid edge effects)

[0123] Negative control group: 200 μL of complete culture medium (cell-free) per well

[0124] Experimental group: 200 μL of culture medium (containing cells) with the test substance per well.

[0125] Statistical requirements: Each group had 6 replicates. Data were expressed as mean ± standard deviation. One-way ANOVA was performed using GraphPad Prism 9.0. *p<0.05 was considered statistically significant.

[0126] The results are as follows Figure 7 As shown, according to the in vitro cytotoxicity test criteria in GB / T 16886.5-2017 for biological evaluation of medical devices, the recombinant type I collagen of the present invention can be identified as non-cytotoxic.

[0127] Example 7: Regulation of AQP3 and Laminin5 gene expression in HaCaT cells by recombinant type I collagen

[0128] HaCaT cells (human keratinocytes, CAS Cell Bank) were prematurely revived and passaged in DMEM medium containing 10% FBS when cell confluence reached 90%. Cells in logarithmic growth phase were cultured at 5 × 10^5 cells per well.

[0129] HaCaT cells were seeded into 6-well plates and then cultured in a static incubator at 37°C and 5% CO2 for 12 hours (at which point cell confluence reached over 80%). After culture, the culture medium was removed, and the cells were washed twice with PBS to remove residual medium. Next, 2 mL of a serum-free medium diluted to a final concentration of 1 μg / mL was added to each well, with PBS used as a control. The cells were then returned to the 37°C, 5% CO2 incubator, and samples were collected after 24 hours. Before sampling, the wells were washed 2-3 times with PBS to remove excess liquid. Then, 0.5 mL of Trizol was added to each well, and the cells were repeatedly aspirated to collect the adherent cells. Finally, all samples were transferred to 1.5 mL RNase-free centrifuge tubes. RNA was extracted using a kit; detailed instructions were provided in the kit's manual (Novizan). Reverse transcription (with an equal volume of RNA) to cDNA was performed followed by qPCR; specific procedures were described in the kit's manual (Aikerui). 2^(-ΔΔCt) is a method for calculating relative expression levels, where ΔΔCt represents the change in the cycle threshold (Ct) after baseline correction. The following are the steps for calculating 2^(-ΔΔCt):

[0130] Calculate ΔCt for a single sample: For each sample, first calculate the Ct difference between its target gene and reference gene.

[0131] The formula is: ΔCt=Ct_Target-Ct_Reference

[0132] Here, Ct_Target is the cycle threshold of the target gene, and Ct_Reference is the cycle threshold of the internal reference gene.

[0133] Calculate the average ΔCt of the control samples: Select a set of control samples (usually calibration samples or blank controls) and calculate the average ΔCt.

[0134] The formula is: ΔCt_mean = mean(ΔCt_controls)

[0135] Calculate ΔCt: For each test sample, calculate the difference between its ΔCt and the average ΔCt of the control samples.

[0136] The formula is: ΔCt=ΔCt-ΔCt_mean

[0137] Calculate the relative expression level (2^(-ΔΔCt)): Finally, use the formula 2^(-ΔΔCt) to calculate the relative expression level of the target gene relative to the control sample. This formula is based on the assumption that the cycle threshold (Ct) is inversely proportional to the initial template amount, and the 2^x function reflects this inverse relationship. This method quantifies the relative expression level of the target gene in different samples. It is important to note that this calculation method assumes that the expression of the internal reference gene is stable in all samples and that the amplification efficiency is the same. In practical applications, these assumptions should be ensured to hold or appropriate corrections should be made.

[0138] The results are as follows Figure 8 As shown, the recombinant type I collagen COL152 of the present invention can significantly upregulate AQP3 and Laminin5 at a concentration of 1 μg / mL (1 ppm), which is beneficial for resisting skin aging and promoting damage repair.

[0139] Example 8: Regulation of collagen and vascular endothelial factor gene expression in HaCaT and HFF-1 cells by recombinant type I collagen.

[0140] Following the method described in Example 7, COL152 was added to pre-coated HaCaT (human keratinocytes, Zhongke Cell Bank) and HFF-1 (human fibroblasts, Zhongke Cell Bank) cells at a final concentration of 5 μg / mL. PBS was used as a control group. RNA was extracted using a kit; detailed steps were provided in the kit's instruction manual (Novizan). After reverse transcription (with the addition of an equal volume of RNA) to cDNA, qPCR was performed; specific procedures were described in the reagent's instruction manual (Aikerui).

[0141] The results are as follows Figure 9 As shown, the recombinant type I collagen of this invention can significantly upregulate the expression of multiple collagen and vascular endothelial factor genes at a concentration of 5 μg / mL (5 ppm). Quantitative analysis results showed that, compared with the control group, the expression of COL1 gene in HFF-1 cells of the experimental group was upregulated by 54%, COL3 gene by 82%, COL4 gene by 130%, COL7 gene by 26%, and VEGF gene by 19%, while the expression of COL7 gene in HaCaT cells of the experimental group was upregulated by 38% and COL17 gene by 79%.

[0142] Example 9: Regulation of matrix metalloproteinase gene expression in HFF-1 and 3T3 cells by recombinant type I collagen.

[0143] The matrix metalloproteinase family (MMP, ADAM) is a key enzyme class that regulates the degradation of the extracellular matrix (ECM). Abnormal expression of these enzymes is directly related to skin photoaging, impaired wound healing, and fibrotic diseases.

[0144] Following the method described in Example 7, COL152 was added to pre-coated HFF-1 (human fibroblasts, CAS Cell Bank) and 3T3 (mouse fibroblasts, Haixing Biotechnology) cells at final concentrations of 5 μg / mL and 190 μg / mL, respectively. PBS was used as a control group. RNA was extracted using the kit; detailed steps were provided in the kit's instruction manual (Novizan). After reverse transcription (with the addition of an equal volume of RNA) to cDNA, qPCR was performed; specific procedures were described in the reagent's instruction manual (Aikerui).

[0145] The results are as follows Figure 10 As shown, the recombinant type I collagen of this invention can significantly downregulate the expression of matrix metalloproteinase genes. This indicates that the recombinant collagen of this invention can regulate the transcription of MMPs, and its mechanism may involve integrin-growth factor receptor crosstalk and oxidative stress inhibition, thereby maintaining the dynamic balance of collagen synthesis / degradation.

[0146] Example 10: Regulation of Inflammatory Factor Expression in HaCaT Cells by Recombinant Type I Collagen

[0147] Recombinant type I collagen regulates the levels of inflammatory factors in HaCaT cells through a multi-level mechanism of "receptor recognition-signal transduction-transcriptional regulation," from gene transcription to protein expression, ultimately affecting the inflammatory state of the cells.

[0148] Following the method described in Example 7, the cell model used was HaCaT cells (human keratinocytes, from the CAS Cell Bank). Cells were cultured and passaged normally, and cells in the logarithmic growth phase (P5-P8 generations) were used for experiments. After trypsin digestion, the cells were resuspended and the density adjusted to 2.5 × 10^5 cells / mL. They were then seeded in 6-well plates, with 2 mL of cell suspension added to each well (i.e., 5 × 10^5 cells / well). After plating, the cells were incubated statically for 12 hours. Microscopic examination confirmed that the adhesion rate was >95% and the morphology was a typical fibroblast-like spindle shape. The cells were washed twice with PBS (1 mL each time, gently aspirated) to remove serum residue and dead cell debris. H2O2 was freshly prepared at 800 μM using serum-free DMEM, with 1 mL of H2O2 solution added to each well, and the cells were cultured for another 4 hours. Experimental grouping and treatment:

[0149]

[0150] Extract RNA using the kit; refer to the kit instructions (Novizan) for detailed steps. Reverse transcribe (add an equal volume of RNA) to cDNA, then perform qPCR; refer to the reagent instructions (Aikerui) for specific procedures.

[0151] The results are as follows Figure 11 As shown, compared with the model group, the recombinant type I collagen of the present invention can significantly downregulate the expression of inflammatory factors IL-1α, IL-8, TNF-α, and COX-2.

[0152] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant type I collagen, characterized in that, Its amino acid sequence is formed by repeated tandem of n basic units, where n is an integer of 1 or greater than 1, and the basic unit contains the Gly-Glu-Pro-Gly-Glu-Pro fragment.

2. The recombinant type I collagen according to claim 1, characterized in that, The number of amino acid residues in the basic unit is less than 50.

3. The recombinant type I collagen according to claim 1, characterized in that, The amino acid sequence of the basic unit is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:

8.

4. The recombinant type I collagen according to claim 1, characterized in that, The value n is selected from any one of the following: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

5. The recombinant type I collagen according to claim 1, characterized in that, The value of n is 8.

6. The gene encoding the recombinant type I collagen as described in claim 1.

7. A recombinant vector expressing the recombinant type I collagen according to claim 1, characterized in that, It contains the gene as described in claim 6.

8. Recombinant cells expressing the recombinant type I collagen of claim 1, characterized in that, The recombinant cells contain the recombinant vector as described in claim 7.

9. The method for preparing recombinant type I collagen according to claim 1, characterized in that, Includes the following steps: 1) The gene described in claim 6 is recombined into the vector pPICZαA to obtain a recombinant vector; 2) The recombinant vector was transfected into Pichia pastoris (Komagataella phaffii) to obtain recombinant cells; 3) After culturing the recombinant cells, centrifuge and collect the supernatant; 4) The recombinant type I collagen was obtained by separating it from the supernatant.

10. The application of the recombinant type I collagen according to claim 1 and the recombinant type I collagen prepared by the preparation method according to claim 9 in the preparation of medical devices or daily chemical products.

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