Recombinant collagen type i with thermostable triple helix structure and its use

Abstract

The application provides a recombinant type I collagen with a heat-stable triple helix structure and an application thereof, and belongs to the technical field of protein engineering, wherein the amino acid sequence of the recombinant type I collagen is shown as SEQ ID NO:1; the application further provides a construction method of an expression system and a preparation method of the recombinant type I collagen; and the application further provides practical industrial applications according to the characteristics of the protein.

Description

Technical Field

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

[0002] Collagen is widely found in tissues such as skin, bone, and muscle. It is a major structural protein in the extracellular matrix of mammalian cells, playing various physiological functions in regulating cell proliferation, differentiation, and migration. Collagen has a triple helix structure, in which three polypeptide chains form a helical coil. Each polypeptide chain consists 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. Due to its excellent biological properties, collagen is widely used in tissue engineering, clinical medicine, the food industry, cosmetics, and medical aesthetics.

[0003] In recent years, collagen has been widely used in the medical field, such as as a component of biocompatible materials for artificial organ grafts, assisting in sustained drug release, artificial skin, and wound care matrices, as well as tissue fillers. Type I collagen is the most abundant form in the human body.

[0004] Currently, much of the collagen used in medical devices is derived from non-human mammal species such as cattle or pigs. Animal-derived collagen presents the following problems: harsh extraction conditions lead to protein structure damage, resulting in poor product uniformity and unstable biological efficacy and clinical results; the raw material source carries the risk of animal-derived pathogen contamination and antibiotic residues; and it possesses a certain degree of immunogenicity, easily triggering an immune response in the body, leading to adverse reactions such as allergies, inflammation, and fever.

[0005] With the rise of the synthetic biology industry, researchers have turned their attention to systems for producing safe human collagen. For example, using mammalian cells, yeast, or E. coli to produce recombinant collagen. However, currently available recombinant collagen does not perform well. Firstly, it lacks the triple helix structure inherent in natural collagen; secondly, it is easily degraded, has poor function, and low yield, making it unsuitable for industrialization. Summary of the Invention

[0006] 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 the characteristics of being able to form a triple helix structure, having high purity, and good thermal stability.

[0007] The recombinant type I collagen of this invention has a basic structure consistent with human type I collagen, and is further optimized to improve its performance. Specifically, the recombinant type I collagen of this invention is obtained by connecting and repeatedly tandemly linking multiple truncated regions. For example, the recombinant collagen of this invention is obtained by truncating 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, 1-3 amino acids, or 1-2 amino acids from human type I collagen.

[0008] The present invention discloses a recombinant type I collagen whose amino acid sequence contains n repeating units, where n is an integer greater than 1, and the repeating units contain the fragment shown in GEEGKRGPPGFPGPAGPRGPPGESGPPGPS (SEQ ID NO: 2).

[0009] The n repeating units are repeated in series, either by connecting the repeating units head to tail or by connecting the repeating units in series by a linker. The linker preferably contains glycine (Gly) and / or serine (Ser) residues, and preferably contains 1-5 amino acid residues.

[0010] Preferably, the amino acid sequence of the recombinant type I collagen is shown in SEQ ID NO: 1.

[0011] The recombinant type I collagen has an apparent relative molecular weight of approximately 22.113 kDa and a theoretical isoelectric point of 6.69.

[0012] 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 repeating unit sequences in the form of single-chain structures still falls within the protection scope of this invention.

[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, so as to construct and express a novel target protein with multiple functions; it can also be linked with tags that help 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 to enhance the stability of the RNA secondary structure, thereby enabling the synthesized peptide chain to better form the target protein and thus 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] Collagen, as a major component of the natural extracellular matrix, provides excellent support for cells through its unique triple helix structure and collagen fibers, creating a suitable microenvironment for cell proliferation and migration. 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 fail to form higher-order structures and lack sufficient mechanical strength, making them easily hydrolyzed by proteases after implantation. Therefore, this invention addresses this issue by optimizing the structure and properties of collagen, such as improving thermal stability to ensure the raw material possesses sufficient mechanical strength and remains "inert" to proteases. For example, designing an appropriate primary structure strengthens the rigidity of the triple helixes in certain regions, resulting in tighter interchain interactions and improved thermal stability.

[0017] 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 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.

[0018] This invention provides a gene encoding the recombinant type I collagen described above.

[0019] 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.

[0020] The present invention provides a recombinant vector expressing the recombinant type I collagen, comprising the aforementioned gene.

[0021] 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 one or more nucleic acid molecules that are operatively linked. 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.

[0022] 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.

[0023] The present invention provides recombinant cells expressing the recombinant type I collagen, wherein the recombinant cells are transfected with the recombinant vector.

[0024] The recombinant cells include, but are not limited to, Escherichia coli, yeast, mammalian cells, and plant cells.

[0025] The Escherichia coli was BL21(DE3).

[0026] The yeast is Pichia pastoris (Pichia pastoris) Komagataella phaffii X-33 or GS115.

[0027] The mammalian cells may be Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK293) cells, or fibrosarcoma cell line HT1080.

[0028] 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.

[0029] A second objective of this invention is to provide a method for expressing the recombinant type I collagen, comprising the following steps: 1) The gene is ligated into a vector to obtain a recombinant vector; 2) Transfect the recombinant vector into cells 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.

[0030] In a preferred embodiment, the recombinant type I collagen is expressed in Pichia pastoris.

[0031] Preferably, in step 2), the gene encoding lysine hydroxylase and / or the gene encoding proline hydroxylase are also transfected.

[0032] Furthermore, the recombinant type I collagen encoding gene can be expressed alone or co-expressed with the viral lysyl hydroxylase L230 gene, proline hydroxylase L593 gene, and / or anthrax P4H gene.

[0033] 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.

[0034] Currently, approximately 29 types of collagen are reported to exist in vertebrates. Based on primary structure, length of triple helix domains, molecular weight, size and shape of triple helix interruptions and terminal domains, collagen can be divided into four major categories: Category 1, fibrous collagen, includes types I, II, III, V, XI, XXIV, and XXVII. Types I, II, and III are more abundant in vertebrates, while types V and VI are less abundant but play an auxiliary role in the assembly of types I, II, and III. Category 2, triple helix fibrous collagen (FACIT), includes types IX, XII, XIV, XVI, XIX, and XXII. FACIT collagen does not form collagen, but it can interact with fibrillary collagen to regulate the formation and size of collagen fibers and control collagen synthesis in the extracellular matrix; the third type is reticular collagen, including types IV, VII, and XXVIII. Type IV collagen forms a fibrous network structure, while type VII collagen assembles into anchoring fibers that connect the epidermis to the dermis; the fourth type is discontinuous helical collagen (MACITs), including types XIII, XXIII, and XXV (Ye et al., 2023).

[0035] Based on this, the recombinant type I collagen of the present invention can be used in combination with one or more of recombinant or natural type I fibrous collagen, type II triple-helix fibrous collagen, type III reticular collagen and type IV discontinuous helix collagen. More preferably, it can be prepared and used by fusing expression with bioactive components such as type III collagen, type IV collagen, type VII collagen, type XVII collagen, fibronectin, and human epidermal growth factor. It can also be used by mixing with the above-mentioned bioactive components after separate preparation.

[0036] Subcutaneous injection filler

[0037] Subcutaneous filler injection is a technique that involves injecting filler material percutaneously into appropriate locations to achieve cosmetic results. Ideal soft tissue fillers should possess good safety, biocompatibility, and effectiveness. Currently, facial fillers primarily utilize 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. Localized facial collagen injections can achieve effects such as facial contour correction, wrinkle reduction, and scar repair.

[0038] Medical repair materials

[0039] 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.

[0040] 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.

[0041] In one specific case, collagen sponge artificial dura mater was applied to patients with post-traumatic epilepsy. It was found that 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).

[0042] Medical transplant materials

[0043] 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's damaged or missing tissue or organ sites. This results in new tissue that closely resembles normal tissue, restoring the damaged shape or some function, thus achieving the goal of regenerating damaged tissues and organs. Therefore, the required biological scaffolds must be biodegradable, possess revascularization capabilities, be able to colonize fibrocartilage tissue, be biocompatible, not produce toxic degradation products, and possess the mechanical properties of tissue for wound healing.

[0044] Type I fibrillary collagen is the main collagen in the heart muscle, accounting for 85% of total collagen, while type III accounts for 11%. Type I collagen or its functional fragments may help treat diseases of heart valves or other areas of the heart, or to create, treat, or maintain transplantable bioequivalents of parts of the heart. Type I collagen may help improve the success rate of such transplants, extend the lifespan of the heart and its valves, or improve the success rate of cardiac surgery.

[0045] 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 create, treat, or maintain transplantable bioequivalents of parts of the eye. Type I collagen can be used to improve the success rate of 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.

[0046] Type I collagen is the most common type of collagen and is essential for the structural integrity of various tissues. It is present in almost all connective tissues and is a major component of the interstitial matrix. It is currently used as a scaffold material in bone tissue engineering methods.

[0047] 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 in periodontal bone defects, accelerating periodontal bone hyperplasia and gingival regeneration. It is also the most abundant matrix protein in bone tissue.

[0048] Daily chemical products

[0049] Compositions containing recombinant collagen can be applied topically to the skin to reduce skin damage or promote the repair of damaged skin. For example, these compositions can protect the skin from UV damage, protect skin cells from the effects of oxidation, promote the repair of damaged skin by increasing cell vitality and / or increasing procollagen synthesis when applied to the skin, and / or promote skin cell vitality. The compositions described herein can compensate for collagen loss associated with aging and skin damage. The compositions described herein can be used as skin moisturizers.

[0050] 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, according to practical applications. The dosage form of the composition includes, but is not limited to, creams, lotions, aqueous solutions, gels, oils, powders, muds, patches, films, or freeze-dried products; furthermore, to promote transdermal absorption of collagen molecules, patch products can be prepared using solid carriers such as non-woven fabrics and applied to the skin surface to prolong the action time of the collagen solution.

[0051] From the perspective of end products, the collagen of the present invention can be applied to daily chemical products. The composition containing recombinant collagen may also contain at least one additional ingredient, which includes other active ingredients, topical carriers and / or preservatives, and may further contain a carrier liquid, stabilizer, preservative, thickener, emulsifier, humectant, emollient and / or other ingredients.

[0052] In various applications, compositions containing recombinant collagen are personal care products that may also include other cosmetic ingredients suitable for human use. These personal care products can be used to prevent or treat damage to human skin or hair caused by external environmental factors. Applications can include skin cleansing and repair products, hair cleansing and repair products, such as face masks, serums, creams, soaps, cleansing pastes, cleansing milks, facial cleansers, shampoos, conditioners, lotions, and shower gels.

[0053] Compared with the prior art, the present invention has the following beneficial effects: 1) The recombinant type I collagen of this 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 optimizes the structurally stable region and functional active site to fully exert biological functional activity. 2) The recombinant type I collagen of this invention has a triple helix structure consistent with natural collagen and has thermal stability higher than human body temperature, which is beneficial to industrialization and downstream applications; it has good biocompatibility and is an excellent material for medical filling. Attached Figure Description

[0054] Figure 1 This is an SDS-PAGE image of the fermentation supernatant of the recombinant type I collagen COL148 of this invention.

[0055] Figure 2 This is an SDS-PAGE image of the purified recombinant type I collagen COL148 of this invention.

[0056] Figure 3 This is a circular dichroism chromatogram of the recombinant type I collagen COL148 of this invention at room temperature.

[0057] Figure 4 The image shows circular dichroisms of the recombinant type I collagen COL148 of this invention at different temperatures.

[0058] Figure 5 The results of the in vitro trypsin digestion test of recombinant type I collagen COL148 of this invention are shown.

[0059] Figure 6 The results of the in vitro collagen enzymatic hydrolysis test of recombinant type I collagen COL148 of this invention are shown. Detailed Implementation

[0060] 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.

[0061] The present invention does not specifically limit the separation and purification method, and conventional protein separation and purification methods in the art can be used; preferred technical solutions are described in the embodiments.

[0062] 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.

[0063] Example 1: Expression, isolation, and purification of recombinant type I collagen of the present invention

[0064] 1. Sequence synthesis

[0065] GenScript Biotech Inc. was commissioned to synthesize the gene fragment of the recombinant type I collagen (referred to as COL148) of this invention and construct it into the pPICZalphaA Pichia pastoris expression vector.

[0066] 2. Construction of Pichia pastoris expression system

[0067] 2.1 Electric Rotor

[0068] 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 subjected to plasmid electroporation. After electroporation, sorbitol was added, and the cells were transferred to sterile centrifuge tubes and activated at 30°C and 230 rpm for 2 h. The cells were centrifuged at 5000g for 1 min, 300 μl of supernatant was discarded, and the cells were resuspended and plated on YPDSZ agar plates (tryptone 20 g / L, yeast extract 10 g / L, D-sorbitol 182.1 g / L, 20% glucose, 100 μg / ml bleomycin) and incubated at 30°C and 230 rpm for 48 h.

[0069] 2.2 Remove the plate containing colonies, pick out single colonies, and copy them onto YPD solid medium containing bleomycin for screening.

[0070] 2.3 Remove the plates containing colonies, pick single colonies, and inoculate them into YPD liquid medium (containing 100 μg / ml bleomycin) for overnight seed culture at 30°C and 230 rpm. Inoculate the overnight seed culture into 1L BMGY medium at a 1:50 ratio for scale-up. Incubate at 30°C and 230 rpm for 48 h. Centrifuge the BMGY culture at 3000 rpm for 5 min, discard the supernatant, resuspend in sterile water, centrifuge again at 3000 rpm for 5 min, discard the supernatant, add 1L BMMY medium (without methanol) for resuspending, and add 1% methanol for induction. After 72 h of induction, centrifuge and collect the supernatant. SDS-PAGE electrophoresis is used to verify the presence of the target protein in the fermentation supernatant. Protein loading buffer (with added DTT) is added to the supernatant sample, mixed well, and placed in a metal bath at 95°C for 10 min before SDS-PAGE analysis. The results are shown below. Figure 1 As shown, the molecular weight of COL148 is approximately 30 kDa, which differs from the theoretical molecular weight of 22.113 kDa. It is speculated that this difference may be due to the fact that collagen is rich in proline / hydroxyproline, which hinders the uniform encapsulation of SDS and affects the linearity of migration.

[0071] 2.4 Ion exchange purification

[0072] The target protein was dialyzed into buffer A (20 mM PB). The protein sample was filtered through a 0.45 μm pore size, 13 mm diameter filter membrane, and cation exchange was performed 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.

[0073] Example 2: Circular dichroism (CD) detection of structural features of recombinant type I collagen

[0074] The recombinant type I collagen lyophilized powder prepared in Example 1 was dissolved in 20 mM PB buffer 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. 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 sample COL148 of the present invention has a triple helix structure.

[0075] 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.

[0076] The RPN values ​​of the recombinant type I collagen sample COL148 of the present invention are shown in Table 1.

[0077] Table 1 COL148 2.34776 30.4832 0.078

[0078] 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, based on the disappearance of the characteristic positive peak at 221 nm, the melting temperature of the recombinant type I collagen sample COL148 of this invention is 42 °C. This indicates that the triple helix structure formed by the protein is tightly wrapped and can better withstand the normal human body temperature (about 36.5 °C), which is beneficial for use as implantation, filling and other related materials.

[0079] Example 4: In vitro anti-degradation experiment to detect the stability of recombinant type I collagen

[0080] The recombinant type I collagen lyophilized powder prepared in Example 1 and commercially available bovine type I collagen (animal-derived extract) were dissolved separately 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 enzyme-to-substrate mass ratio 1:200. The mixtures were incubated in a 37°C water bath, and samples were taken at time points of 0 min, 10 min, 20 min, 30 min, 60 min, 2 h, 4 h, 8 h, 24 h, and 48 h. 20 μL of the reaction solution was taken, and protein loading buffer containing DTT was added. The mixture was immediately vortexed and mixed. The sample was heated in a metal bath at 95°C 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 used for SDS-PAGE electrophoresis (conditions: constant voltage 160 V, electrophoresis time 40 min). Observe the band distribution after Coomassie brilliant blue staining.

[0081] like Figure 5 and 6 As shown, the recombinant type I collagen COL148 of this invention still showed a clear target protein band in the SDS-PAGE results after 1 hour of digestion with Trypsin and collagenase, while the commercially available bovine type I collagen control was degraded after 10 minutes, and the SDS-PAGE results showed no target protein band. The results indicate that the recombinant collagen prepared by this invention has a certain resistance to enzymatic degradation compared to animal-derived products, making it suitable for biomedical applications requiring long-term stability (such as tissue engineering scaffolds or sustained-release formulations).

[0082] 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 having a heat-stable triple helix structure, characterized in that, The amino acid sequence of the recombinant type I collagen is shown in SEQ ID NO:

1.

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

3. A recombinant vector expressing the recombinant type I collagen according to claim 1, characterized in that, It contains the gene described in claim 2.

4. 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 3.

5. 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 2 is recombined into a vector to obtain a recombinant vector; 2) Transfect the recombinant vector into cells 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.

6. The preparation method according to claim 5, characterized in that, In step 2), the gene encoding lysine hydroxylase and / or the gene encoding proline hydroxylase were also transfected.

7. 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 5 in the preparation of medical devices.

8. The application according to claim 7, characterized in that, The recombinant type I collagen is used to prepare therapeutic biocompatible materials.

9. The application according to claim 7, characterized in that, The recombinant type I collagen is used to prepare micro-plastic surgery filler materials.

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 5 in the preparation of daily chemical products.

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