Recombinant I-type collagen with thermostable triple-helix structure and application of recombinant I-type collagen

Abstract

The invention provides a recombinant I-type collagen with a thermostable 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 shown as SEQ ID NO: 1; 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.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and in particular relates to recombinant type I collagen with a heat-stable triple helical structure and applications thereof. Background Art

[0002] Collagen is widely present in tissues such as skin, bone, and muscle. It is the main structural protein in the extracellular matrix of mammals and has various physiological functions in regulating cell proliferation, differentiation, and migration. The structure of collagen is a triple helix, in which three polypeptide chains together form a spiral coil. Individual polypeptide chains are composed of a repeating triplet amino acid sequence of Gly-XY, where X and Y can be any amino acids. The most common triplet is proline-hydroxyproline-glycine (Gly-Pro-Hyp), which accounts for approximately 10.5% of collagen triplets. Collagen is widely used in tissue engineering, clinical medicine, food industry, cosmetics, and medical cosmetology due to its excellent biological properties.

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

[0004] Much of the collagen currently used in medical devices comes from non-human mammalian species, such as cows or pigs. Animal-derived collagen presents several challenges: harsh extraction methods can disrupt protein structure, leading to poor product uniformity and unstable biological efficacy and clinical results. Raw material sources can also harbor the potential for contamination with animal pathogens and antibiotic residues. Furthermore, animal-derived collagen can be immunogenic, potentially triggering immune responses and causing adverse reactions such as allergies, inflammation, and fever.

[0005] With the rise of the synthetic biology industry, researchers are turning their attention to systems for producing safe human collagen. For example, recombinant collagen can be produced using mammalian cells, yeast, or E. coli. However, currently available recombinant collagen is not very effective. First, it lacks the triple helical structure inherent in natural collagen. Second, it is easily degraded, has poor functionality, and has low yields, making it unsuitable for industrialization. Summary of the Invention

[0006] In view of this, the first object of the present invention is to provide a recombinant type I collagen with a triple helical structure. The recombinant collagen provided by the present invention has the characteristics of being able to form a triple helical structure, high purity, and good thermal stability.

[0007] The recombinant type I collagen of the present invention maintains the same basic structure as human type I collagen, while also being optimized to enhance its performance. 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 by 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.

[0008] The recombinant type I collagen described in the present invention has an amino acid sequence comprising n repeating units, wherein n is an integer of 1 or greater than 1, and the repeating unit comprises a fragment represented by GEEGKRGPPGFPGPAGPRGPPGESGPPGPS (SEQ ID NO: 2).

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

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

[0011] The apparent relative molecular weight of the recombinant type I collagen is approximately 22.113 KDa, and the theoretical isoelectric point is 6.69.

[0012] It should be understood that the present invention essentially protects the amino acid sequence of recombinant type I collagen. Therefore, the large-scale production of proteins containing repeating unit sequences in the form of single-chain structures still falls within the scope of protection of the present invention.

[0013] In the present invention, the recombinant type I collagen can be used as a structural domain to connect with structural domains from other sources to form a fusion protein to construct and express a new target protein with multiple functions; it can also be connected to a tag that helps protein expression, purification or detection.

[0014] In the present invention, the introduction of amino acids at the N-terminus of the protein can refer to the method described in CN117511978A to enhance the stability of the RNA secondary structure, so that the synthetic peptide chain can better form the target protein, thereby increasing the yield of the exogenous protein.

[0015] In the present invention, the amino acids introduced into the C-terminus of the protein may be amino acids derived from human type I collagen.

[0016] Collagen is as the main component of natural extracellular matrix, and its unique triple helical structure and collagen fibers play a good supporting role to cells, for the proliferation of cells, migration etc. create suitable microenvironment.But at present animal source is because of the introduction of complicated or too many chemical reagents of extraction method, the original natural cross-linked structure of collagen can be destroyed in varying degrees, and many commercially available recombinant collagens can not form high-order structure equally, lack enough mechanical strength, are easy to be hydrolyzed by protease in vivo in implantation.Therefore the present invention considers application, also collagen is carried out structure and performance optimization, for example, improves thermostability, to ensure that collagen material has certain mechanical strength and maintenance " inertia " to protease.For example, design appropriate primary structure, make the triple helix rigidity that part area forms strengthen, interchain action is tighter, improves thermostability.

[0017] The present 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 of left- and right-handed optical rotations, primarily for determining molecular structural asymmetry. Biomacromolecules generally contain chiral groups and structures, and therefore CD is often used to measure and observe structural and conformational changes in these molecules. The CD characteristic of collagen's triple helix structure is generally characterized by 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 amino acid sequence and length. The thermal stability of collagen includes the thermal shrinkage temperature (Ts) and thermal denaturation temperature (Td) of collagen fibers. The thermal shrinkage temperature of collagen fibers refers to the temperature at which the fibers undergo axial contraction when heated. The thermal denaturation temperature of collagen refers to the temperature at which, when heated in a medium, the triple helix unwinds to form single strands, reaching 50% of its original value. Therefore, CD spectroscopy can be used to study the helical structure of collagen and its thermal denaturation process.

[0018] The present invention provides a gene encoding the recombinant type I collagen.

[0019] The encoding includes a protein produced by transcribing a DNA molecule to form an RNA product, which is then translated; or a protein produced by transcribing a DNA molecule to provide an RNA product, which is processed to provide a processed RNA product, which is then translated.

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

[0021] Vectors include any nucleic acid molecule derived from any source and capable of genomic integration or autonomous replication (e.g., a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule), comprising one or more operably linked 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, such vectors include, but are not limited to, pPICZαA and pPIC9k.

[0022] In the present invention, the recombinant human type I collagen gene may be cloned into the vector alone, or the proline hydroxylase gene and / or lysine hydroxylase gene and the recombinant human type I collagen gene may be cloned into the same vector.

[0023] The present invention provides a recombinant cell expressing the recombinant type I collagen, wherein the recombinant cell is 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 is BL21 (DE3).

[0026] The yeast is 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 vectors into other host cells are known, and similar methods can be used in the present invention.

[0029] The second object of the present invention is to provide a method for expressing the recombinant type I collagen, comprising the following steps: 1) connecting the gene to a vector to obtain a recombinant vector; 2) transfecting the recombinant vector into cells to obtain recombinant cells; 3) culturing the recombinant cells, centrifuging, and collecting the supernatant; 4) Separating and obtaining the recombinant type I collagen from the supernatant.

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

[0031] Preferably, in step 2), a gene encoding lysine hydroxylase and / or a gene encoding proline hydroxylase is 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 Bacillus anthracis P4H gene.

[0033] The third object of the present invention is to provide the use of the recombinant type I collagen and the recombinant type I collagen prepared by the preparation method in the preparation of medical devices or daily chemical products.

[0034] Currently, there are approximately 29 types of collagen in vertebrates. Based on primary structure, triple helical domain length, molecular weight, triple helical interruptions, and the size and shape of the terminal domains, collagen can be divided into four major categories: The first category is fibrillar collagen, which includes types I, II, III, V, XI, XXIV, and XXVII. Types I, II, and III are more prevalent in vertebrates, while types V and VI are less common but play a supporting role in the assembly of types I, II, and III. The second category is fibrillar triple helical collagen (FACIT), which includes types IX, XII, XIV, XVI, XIX, and XXII. FACIT collagens do not form collagen, but can interact with fibrillar collagens, regulate the formation and size of collagen fibers, and control collagen synthesis in the extracellular matrix. The third type of reticular collagens includes types IV, VII, and XXVIII. Type IV collagen forms a fibrillar network structure, while type VII collagen assembles into anchoring fibers that connect the epidermis to the dermis. The fourth type of interrupted helical collagens (MACITs) includes types XIII, XXIII, and XXV (Ye Tao 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 1 fibrillar collagen, type 2 triple helical fibrillar collagen, type 3 reticular collagen and type 4 interrupted helical collagen. More preferably, it is prepared and used by fusion expression with biologically active 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 mixed with the above-mentioned biologically active components after being prepared separately.

[0036] Subcutaneous injection filler materials

[0037] Subcutaneous injection filler technology uses percutaneous injection to inject filler materials into appropriate locations in a targeted, targeted, and quantitative manner to achieve the desired cosmetic effect. Ideal soft tissue filler materials should possess excellent safety, biocompatibility, and effectiveness. Currently, facial micro-plastic surgery fillers are primarily non-autologous tissue injectables, such as hyaluronic acid (HA), collagen, and other single-ingredient fillers. Collagen is widely used in medical aesthetics to repair skin defects and subcutaneous diseases. Local facial collagen injections can achieve effects such as facial contour correction, wrinkle reduction, and scar repair.

[0038] Medical restorative materials

[0039] Collagen materials have a natural porous network structure. When in contact with the wound, they can quickly absorb blood, promote platelet aggregation, and absorb tissue permeate, creating a good wound microenvironment and promoting wound healing. Currently, there are many collagen-based soft tissue repair products in clinical practice, mainly including collagen dressings, dura mater patches, artificial corneas, oral patches, hernia patches, breast plastic surgery patches, etc. Their uses 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 comprises identifying tissue in need of repair, such as collagenous tissue, such as the cornea, skin, bone, connective tissue, such as cartilage, ligament, tendon, or basement membrane; and contacting the tissue with an agent that increases type I collagen. The wound may be caused by trauma (e.g., a tear in connective tissue or muscle), an incision, or surgery (e.g., a transplant), such as a heart transplant, lens transplant, joint replacement, hair follicle replacement, or skin transplant. In one scenario, the method is used to repair a wound in the eye, such as a surgical incision, corneal transplant, LASIK flap reattachment, cataract surgery, laser surgery, keratoplasty, penetrating keratoplasty, posterior lamellar keratoplasty, refractive surgery, corneal reshaping, or treatment of a corneal tear. For another example, the wound is in cardiac tissue, a heart valve, or more specifically, as the result of a heart valve replacement or transplant. For another example, the wound is in cartilage, tendon, or ligament. For another example, the wound is in epithelial tissue, such as the dermis.

[0041] In one specific example, 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, did not increase the incidence of epilepsy in patients, and no patients developed infection or elimination reactions after surgery, indicating that it has good tissue compatibility and can be used for intracranial tumors (including gliomas, meningiomas, pituitary tumors, neurothema and other congenital tumors, metastatic cancer, etc.), intracranial aneurysms and vascular malformations, intraspinal tumors, and other pathological germ cell tumors, epileptic lesions, 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 study of biological substitutes. Using seed cells, scaffolds, and growth factors, tissues and organs similar to those of the human body are constructed and transplanted back into the host's damaged or missing tissues and organs, forming new tissues that resemble normal tissue, restoring the appearance or partial function of the defect, and ultimately regenerating the defective tissue and organ. Therefore, the desired biological scaffolds must be biodegradable, have revascularization capabilities, be able to colonize with fibrocartilage tissue, be biocompatible, not degrade into toxic products, and possess the mechanical properties of tissue for wound healing.

[0044] Type I fibrillar collagen is the predominant collagen in the myocardium, comprising 85% of total collagen, while type III accounts for 11% of total myocardial collagen. Type I collagen or its functional fragments may be useful in treating diseases of heart valves or other areas of the heart, or in creating, treating, or maintaining transplantable bioequivalents of cardiac parts. Type I collagen may help improve the success of such transplants, extend the lifespan of the heart and its valves, or enhance the success of cardiac surgery.

[0045] Because type I collagen is expressed in the muscle-connective tissue border of the eye, type I collagen or its functional fragments may be useful for treating ocular diseases or creating, treating, or maintaining a portion of the eye that can be transplanted into the eye. Type I collagen may be used to improve the success of such transplants, such as lens replacements, or extend the lifespan of the lens and its tissues, or to increase the success of ophthalmic surgeries, such as treating surgically induced endophthalmitis.

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

[0047] Mature dentin consists of approximately 70% hydroxyapatite, 18% collagen, 2% non-collagenous proteins, and 10% water. Collin I (COL I) is the scaffolding structure of the dentin matrix. Combining collagen with hydroxyapatite to form a hydroxyapatite-mineralized collagen complex can be used as an osteoconductive coating and scaffold. For example, in restorative dental treatment, collagen can be added to periodontal bone defects to accelerate periodontal bone growth 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, the compositions can protect the skin from UV damage, protect skin cells from the effects of oxidative exposure, and promote the repair of damaged skin by increasing cell viability and / or increasing procollagen synthesis when applied to the skin, and / or promote the viability of skin cells. 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 a cosmetically or pharmaceutically effective amount of at least one protein according to the aforementioned method and at least one excipient or cosmetically or pharmaceutically acceptable adjuvant according to practical application. 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 forms. Furthermore, in order to promote transdermal absorption of collagen molecules, a solid carrier such as a non-woven fabric can be used to prepare a patch product, which can be applied to the skin surface to prolong the effect of the collagen solution.

[0051] From the perspective of end products, the collagen of the present invention can be used in daily chemical products. The composition containing recombinant collagen can also contain at least one additional ingredient, which includes other active ingredients, topical carriers and / or preservatives, and can further contain carriers, stabilizers, preservatives, thickeners, emulsifiers, wetting agents, emollients and / or other ingredients.

[0052] In various applications, the composition containing recombinant collagen is a personal care product that may contain other cosmetic ingredients suitable for human use. Such personal care products can be used to prevent or treat damage to human skin or hair caused by the external environment. Applications include skin cleansing and repair products, hair cleansing and repair products, such as facial masks, serums, creams, soaps, cleansing pastes, cleansing milks, cleansing milks, shampoos, conditioners, skin care lotions, and body washes.

[0053] Compared with the prior art, the present invention has the following beneficial effects: 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 lower than that of natural full-length collagen (300kDa). The structural stabilization region and functional active sites are optimized to fully exert biological functional activity. 2) The recombinant type I collagen of the present invention has a triple helix structure consistent with natural collagen, and its thermal stability is higher than human body temperature, which is conducive to industrialization and downstream applications; it has good biocompatibility and is an excellent material for medical filling. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0055] Figure 2 This is an SDS-PAGE diagram of the purification of the recombinant type I collagen COL148 of the present invention.

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

[0057] Figure 4 Circular dichroism spectra of the recombinant type I collagen COL148 of the present invention at different temperatures.

[0058] Figure 5 These are the results of an in vitro trypsin hydrolysis test of the recombinant type I collagen COL148 of the present invention.

[0059] Figure 6 This is the result of in vitro collagenase hydrolysis test of the recombinant type I collagen COL148 of the present invention. DETAILED DESCRIPTION

[0060] In the present invention, the gene can be synthesized by a biotechnology company. The present invention has no particular limitation on the preparation method of the recombinant vector and recombinant cell, and conventional preparation methods of recombinant vectors and recombinant cells in the art can be used.

[0061] The present invention has no particular limitation on the separation and purification method, and conventional protein separation and purification methods in the art may be used; for preferred technical solutions, please refer to the examples.

[0062] The technical solutions provided by the present invention are 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 and purification of recombinant type I collagen of the present invention

[0064] 1. Sequence synthesis

[0065] GenScript Biotech Co., Ltd. was commissioned to synthesize the gene fragments of the recombinant type I collagen (abbreviated as COL148) of the present invention and construct them into the pPICZalphaA Pichia pastoris expression vector.

[0066] 2. Construction of Pichia pastoris expression system

[0067] 2.1 Electroporation

[0068] Plasmid pPICZalphaA was linearized at the SacI site by PCR amplification, and the PCR fragment was purified using the ThermoGeneJET PCR Purification Kit. Competent GS115 cells were thawed on ice and electroporated with the plasmid. After electroporation, sorbitol was added, and the cells were transferred to a sterile centrifuge tube and activated at 30°C, 230 rpm, for 2 h. The cells were centrifuged at 5000 g for 1 min, and 300 μl of the supernatant was discarded. The cells were resuspended and plated on YPDSZ plates (20 g / L tryptone, 10 g / L yeast extract, 182.1 g / L D-sorbitol, 20% glucose, and 100 μg / ml bleomycin) and incubated at 30°C, 230 rpm, for 48 h.

[0069] 2.2 Remove the plate with colonies, pick out single clones from it, and replicate them on YPD solid medium with bleomycin for screening.

[0070] 2.3 Take out the plate with colonies, pick out monoclonal colonies, and inoculate them into YPD liquid medium (containing 100μg / ml bleomycin) for overnight seed culture at 30℃ and 230rpm. Inoculate the overnight cultured seed liquid into 1L BMGY medium at a ratio of 1:50 for amplification. Culture at 30℃ and 230rpm for 48h. Centrifuge the BMGY cultured seed liquid at 3000rpm for 5min, discard the supernatant, resuspend with sterile water, centrifuge again at 3000rpm for 5min, discard the supernatant, add 1L BMMY medium (without methanol) to resuspend, and add 1% methanol for induction. After 72h of induction, centrifuge and collect the supernatant. SDS-PAGE electrophoresis verifies whether the fermentation supernatant contains the target protein. Take the supernatant sample and add Protein loading buffer (add DTT), mix well, place in a metal bath, 95℃, heat for 10min, and then perform SDS-PAGE detection. The results are as follows Figure 1 As shown in the figure, the molecular weight of COL148 is about 30KDa, which is different from the theoretical molecular weight of 22.113KDa. The reason may be that collagen is rich in proline / hydroxyproline, which hinders the uniform packaging of SDS and affects the migration linearity.

[0071] 2.4 Ion exchange purification

[0072] The target protein was dialyzed into buffer A (20mM PB). The protein sample was filtered using a 0.45μm pore size, 13mm diameter membrane filter. Cation exchange was performed using AKTApure (5ml SP column). Buffer B (20mM PB and 1M NaCl, pH = 7.4) was used for linear elution to obtain the target protein with high purity. SDS-PAGE electrophoresis was used to verify the molecular weight and purity of the protein. The protein sample was added to the protein loading buffer (with DTT added), mixed, and placed in a metal bath at 95°C for 10 minutes. After centrifugation at 1500rpm for 1 minute, the supernatant was collected for SDS-PAGE analysis. The results are as follows: Figure 2 As shown. Determine protein concentration using the BCA method to estimate protein yield. Depending on protein purity, concentrate, exchange, and freeze-dry for storage.

[0073] Example 2 Circular Dichroism (CD) Detection of Structural Characteristics 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 spectrometer. The scanning wavelength range was set to 190 nm-260 nm, the scanning speed was set to 100 nm / min, and the scanning temperature was set to room temperature. The CD spectra were all averaged over three scans. The results are shown in Figure 2. 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. According to the circular dichroism characteristics of the known collagen triple helix structure, the recombinant type I collagen sample COL148 of the present invention has a triple helix structure.

[0075] The 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 positive peak intensity to the absolute value of the negative peak intensity: RPN = positive peak intensity / |negative peak intensity|. Changes in the RPN value can, to a certain extent, reflect changes in collagen structure. When the triple helix structure of collagen is significantly disrupted, the RPN value drops to 0 or even becomes negative. For example, the RPN value of completely denatured collagen or gelatin is 0 or even negative. During heating, the triple helix structure of collagen is affected to some extent, leading to changes in the RPN value. This is primarily due to the impact of increased temperature on the hydrogen bonds within the collagen molecule, which in turn affects 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, it is possible to understand the structural stability of collagen under different conditions, such as heating and irradiation.

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

[0077] Table 1 Recombinant type I collagen Positive peak intensity Negative peak intensity RPN 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 detected by real-time temperature increase at a rate of 1°C / min. The CD spectra were the average of three scans, and the data were analyzed and calculated on the software. 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 the present invention is 42°C, indicating that the triple helix structure formed by the protein is tightly wound and can well withstand the normal body temperature of the human body (about 36.5°C), which is conducive to use as related materials such as implants and fillings.

[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) were dissolved in PBS buffer to prepare a 1.0 mg / mL solution. The solution was then allowed to equilibrate to room temperature. Trypsin (purchased from Promega) and collagenase (purchased from Beyotime) were added to each collagen solution to a mass ratio of 1:200. The mixture was incubated in a 37°C water bath, and samples were taken at 0 min, 10 min, 20 min, 30 min, 60 min, 2 h, 4 h, 8 h, 24 h, and 48 h. Twenty μL of the reaction solution was added to protein loading buffer containing DTT and immediately vortexed to mix. The sample was heated in a metal bath at 95°C for 10 min, then centrifuged at 1500 rpm for 1 min. The supernatant was collected and used for later use. 10 μL of the supernatant was subjected to SDS-PAGE electrophoresis (consistent voltage: 160 V, electrophoresis time: 40 min). The band distribution was observed after Coomassie brilliant blue staining.

[0081] like Figure 5 and 6 As shown, the recombinant type I collagen COL148 prepared in this invention still exhibited clear target protein bands on SDS-PAGE after 1 hour of digestion with trypsin and collagenase, whereas a commercially available bovine type I collagen control had degraded after 10 minutes, with no target protein bands observed on SDS-PAGE. These results demonstrate that the recombinant collagen prepared in this invention exhibits a degree of 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 is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A recombinant type I collagen with a heat-stable triple helical structure, characterized in that: The amino acid sequence of the recombinant type I collagen is shown in SEQ ID NO:

1.

2. A gene encoding the recombinant type I collagen according to claim 1.

3. The recombinant vector expressing the recombinant type I collagen according to claim 1, characterized in that: Comprising the gene according to claim 5.

4. The recombinant cell expressing the recombinant type I collagen according to claim 1, characterized in that: The recombinant cell contains the recombinant vector according to claim 3.

5. The method for preparing recombinant type I collagen according to claim 1, characterized in that: The following steps are involved: 1) Recombining the gene according to claim 2 into a vector to obtain a recombinant vector; 2) transfecting the recombinant vector into cells to obtain recombinant cells; 3) culturing the recombinant cells, centrifuging, and collecting the supernatant; 4) Separating and obtaining the recombinant type I collagen from the supernatant.

6. The use according to claim 5, characterized in that In step 2), a gene encoding lysine hydroxylase and / or a gene encoding proline hydroxylase is also transfected.

7. Use of the recombinant type I collagen according to claim 1 or the recombinant type I collagen prepared by the preparation method according to claim 5 in the preparation of medical devices.

8. The use according to claim 7, characterized in that The recombinant type I collagen is used for preparing biocompatible materials for treatment.

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

10. Use of the recombinant type I collagen according to claim 1 or 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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