Biosynthetic type i collagen-like hydrogel for fracture repair and preparation method therefor

Abstract

The present invention belongs to the technical field of genetic engineering. Provided are a biosynthetic type I collagen-like hydrogel for fracture repair and a preparation method therefor. By means of fusion expression of (PPG)10 at the N-terminus and C-terminus of collagen, highly ordered self-assembly of collagen is promoted to form type I collagen-like fibrils with periodic alternating light and dark banding patterns. Moreover, by means of site-directed introduction of Cys into (PPG)10 and the CL-domain, further covalent cross-linking of collagen fibrils is promoted to form a hydrogel. The collagen fibrillar structure within the prepared hydrogel is analogous to that of native type I collagen. The hydrogel has good biocompatibility and bone repair capability, and features a simple preparation process, which enables large-scale production of type I collagen-like fibril hydrogels at a low cost. Thus, the hydrogel has broad application prospects in the field of biomaterials.

Description

A biosynthetic type I collagen hydrogel for fracture repair and its preparation method Technical Field

[0001] This invention relates to a biosynthetic type I collagen hydrogel for fracture repair and its preparation method, belonging to the field of genetic engineering technology. Background Technology

[0002] Collagen, composed of three polypeptide chains forming a triple helix structure, is the most abundant protein in mammals, accounting for one-third of the total protein in the human body. It is a major component of the extracellular matrix and plays a crucial role in regulating cell proliferation and differentiation, as well as in the repair and regeneration of tissues and organs. There are 28 types in mammals, with type I being the most common. In higher organism cells, type I collagen undergoes translation, modification, folding, and cleavage to form regularly arranged banded fibrous structures. This banded feature, characterized by alternating dark and light bands, is known to be essential for osteoblast differentiation and matrix mineralization. This structure facilitates cell adhesion and growth, and plays a key role in the repair and regeneration of tissues and organs. Under an electron microscope, type I collagen fibers exhibit alternating light and dark striped patterns. The unique periodic pattern in collagen is formed by the offset and parallel alignment of the collagen triple helix, which is crucial for cell activity, osteoblast differentiation, and matrix mineralization.

[0003] Type I collagen extracted from animals has wide applications as a biomaterial in food, cosmetics, drug delivery, and biomedical engineering. It can be used in cell culture, tissue engineering, and regenerative medicine. It can also serve as a scaffold or matrix material for skin burn dressings, surgical and dental hemostatic sponges, and bone defect fillings, promoting tissue regeneration and repair. Market demand is huge. Currently, most type I collagen on the market comes from animal skin and connective tissues such as Achilles tendons. While it has the advantages of high biocompatibility and easy absorption by the human body, it is susceptible to contamination from animal-derived diseases such as prions. To improve biosafety, how to prepare clean-source type I collagen is an important issue in the field of biomedical materials.

[0004] Currently, there are four main sources of collagen: 1. Animal extraction: inexpensive, but prone to carrying pathogens; 2. Chemical synthesis: high purity and strong controllability, but expensive, limited in length, and unsuitable for mass production; 3. Expression in eukaryotic systems such as transgenic plants and mammalian cells: capable of correctly folding proteins and performing complex post-translational processing, but with high culture costs, long cycles, low expression levels, and difficulty in large-scale production; 4. Microbial expression systems: with significant advantages such as low cost and high expression levels.

[0005] Studies have shown that an increasing number of mammalian and bacterial collagens are efficiently heterologously expressed in hosts such as bacteria and yeast, and correctly fold into collagen triple helix structures, exhibiting advantages such as low cost and high expression levels. However, most recombinant collagens still lack self-assembly driving forces and cannot polymerize into hydrogels with periodic stripes or specific structures similar to natural type I collagen fibers. This limits their inherent advantages in cell viability, osteogenic differentiation, and biomineralization, hindering their potential applications in biomaterials and tissue engineering.

[0006] Collagen hydrogels are biodegradable, water-rich three-dimensional matrices that provide biological signals and act as cell scaffolds, creating the necessary microenvironment for cell growth, differentiation, and migration, making them suitable for bone repair. Therefore, the preparation of collagen hydrogels with visible collagen streaks and good physiological activity has extremely high practical and economic value. Summary of the Invention

[0007] This application describes the expression of PPG through N-terminal and C-terminal fusion of collagen from different sources. 10 It promotes the high-polymer self-assembly of collagen to form collagen fibers, and through (PPG) 10 Introducing Cys into the neutralized CL-domain promotes further covalent cross-linking of collagen fibers to form a hydrogel.

[0008] The first object of the present invention is to provide a collagen single chain having the following structure:

[0009] From the N-terminus to the C-terminus are: the first repeat sequence, the collagen domain, and the second repeat sequence;

[0010] Specifically, 2 to 5 cysteine ​​residues are introduced into the repeating sequences and / or collagen domains of collagen single chains;

[0011] The introduction is the insertion of cysteine, or the replacement of an amino acid with cysteine.

[0012] Optionally, 2 to 5 cysteine ​​residues may be introduced into the repeating sequence and collagen domain of the collagen single chain.

[0013] In one embodiment, one cysteine ​​residue (named CC) is introduced at the front and the end of the second repeat sequence, respectively.

[0014] Alternatively, one cysteine ​​residue (named NC) may be introduced at the end of the first repeat sequence and the second repeat sequence, respectively.

[0015] In one embodiment, 1 to 3 cysteine ​​residues are introduced into the collagen domain;

[0016] Optionally, one cysteine ​​residue is introduced into the collagen domain;

[0017] Optionally, two cysteine ​​residues are introduced into the collagen domain, with the two cysteine ​​residues separated by 40 to 80 amino acids;

[0018] Optionally, three cysteine ​​residues are introduced into the collagen domain, with the three cysteine ​​residues located in amino acids 1-40, 41-80, and 81-120 of the collagen domain, respectively.

[0019] In one embodiment, the collagen single chain has the following structure:

[0020] The sequence from N-terminus to C-terminus is the first repeat sequence (PPG). n Collagen domain (CL-domain) and second repeat sequence (PPG)n.

[0021] In one embodiment, a folding domain is attached to the N-terminus of the first repeat sequence, and the amino acid sequence of the folding domain is shown in SEQ ID NO.1;

[0022] Optionally, the front of the folded field also includes a 6×His tag.

[0023] In one embodiment, a folding domain is attached to the N-terminus of the first repeating sequence, the amino acid sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.16.

[0024] There is a linker peptide between the folded domain and the repeat sequence, the amino acid sequence of which is shown in LVPRGSPG (SEQ ID NO.4);

[0025] Optionally, the first and second repeat sequences (PPG) n n is an integer from 4 to 30; preferably, n is from 5 to 10; preferably, the amino acid sequences of the first and second repeating sequences are as shown in SEQ ID NO.2;

[0026] Optionally, the collagen domain (CL-domain) includes human collagen, bacterial collagen, recombinant human collagen, and recombinant bacterial collagen;

[0027] Optionally, the amino acid sequence of the collagen domain is shown in SEQ ID NO.3.

[0028] In one embodiment, the structure of a collagen single chain is as follows:

[0029] Among them, the repeating sequence located at the N end (front end) of the CL-domain is the first repeating sequence, and the repeating sequence located at the C end (back end) of the CL-domain is the second repeating sequence.

[0030] In one embodiment, the collagen single chain is as shown in any one of SEQ ID NO. 8 to SEQ ID NO. 11; the folded and enzyme-digested collagen single chain is as shown in any one of SEQ ID NO. 18 to SEQ ID NO. 21.

[0031] In one embodiment, an amino acid is replaced with C at the end of the CL-domain, and a cysteine ​​residue is inserted at the end of the second repeat sequence.

[0032] Optionally, the Q (glutamine) at position 119 of the CL-domain (SEQ ID NO.3) is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, with the amino acid sequence of the collagen single chain as shown in SEQ ID NO.8.

[0033] In one implementation, an amino acid is replaced with C at the front end of the CL-domain, and a cysteine ​​residue is inserted at the end of the second repeat sequence.

[0034] Optionally, the first position (N-terminus) P of the CL-domain (SEQ ID NO.3) is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, the amino acid sequence of the collagen single chain is shown in SEQ ID NO.9.

[0035] In one embodiment, the first P in the CL-domain (SEQ ID NO.3) is replaced with C, and based on the insertion of a cysteine ​​residue at the end of the second repeat sequence (i.e., based on the collagen single chain with the amino acid sequence as shown in SEQ ID NO.9), the amino acid (X or Y in GXY) in the CL-domain is replaced with C.

[0036] Optionally, in the first 1 / 3 of the peptide in the CL-domain (amino acids 1 to 40 of the CL-domain), one X or Y is replaced with C;

[0037] Optionally, in the middle 1 / 3 of the peptide in the CL-domain (amino acids 41 to 80 of the CL-domain), one X or Y is replaced with C;

[0038] Optionally, in the last third of the peptide segment of the CL-domain (amino acids 81 to 120 of the CL-domain), one X or Y is replaced with C.

[0039] In one implementation, the first P in the CL-domain is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, and two X or Y residues in the CL-domain are replaced with C.

[0040] Optionally, in the first half of the peptide in the CL-domain (amino acids 1 to 60 of the CL-domain), one X or Y is replaced with C, and in the last half of the peptide in the CL-domain (amino acids 61 to 120 of the CL-domain), one X or Y is replaced with C.

[0041] Optionally, the distance between the two substituted Cs is 40 to 80 amino acids.

[0042] In one implementation, the first P in the CL-domain is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, and three X or Y residues in the CL-domain are replaced with C.

[0043] Optionally, in the first 1 / 3 of the peptide segment (amino acids 1 to 40 of the CL-domain), the middle 1 / 3 of the peptide segment (amino acids 41 to 80 of the CL-domain), and the last 1 / 3 of the peptide segment (amino acids 81 to 120 of the CL-domain), one X or Y is replaced with C.

[0044] Optionally, in the three substituted Cs, the distance between each pair is 20 to 40 amino acids.

[0045] A second object of the present invention is to provide a gene encoding any of the above-mentioned single chains of collagen, a plasmid or cell carrying the gene encoding any of the above-mentioned single chains of collagen;

[0046] Optionally, the plasmids include the pColdIII series or the pET series.

[0047] In one embodiment, the cells are Escherichia coli cells, including but not limited to E. coli BL21 and E. coli BL21(DE3).

[0048] A third object of the present invention is to provide a collagen product obtained by preparing any of the above-mentioned collagen single chains;

[0049] Optionally, the collagen product includes type I collagen, collagen fibers, and collagen hydrogel.

[0050] In one embodiment, the type I collagen is formed by the high-polymer self-assembly of the above-mentioned collagen single chains, and has a striped pattern of alternating light and dark.

[0051] In one embodiment, the collagen fibers are formed by the high-polymer self-assembly of the aforementioned type I collagen.

[0052] In one embodiment, the collagen hydrogel is prepared by:

[0053] Collagen prepared by fermentation using the above-mentioned collagen gene, plasmid carrying the gene encoding any of the above-mentioned collagen, or cells, is purified, dialyzed, and freeze-dried.

[0054] The freeze-dried collagen was prepared into a solution with a concentration of 0.1–1 mmol / L and allowed to stand at 4℃–37℃ for at least 2 days to obtain type I collagen fibers.

[0055] After freeze-drying, the collagen is prepared into a solution with a concentration of ≥100g / L and left to stand at 4℃~37℃ for 1 week, or hydrogen peroxide is added and left to stand at room temperature for more than 2 hours to obtain type I collagen hydrogel.

[0056] In one embodiment, the type I collagen hydrogel exhibits excellent fracture repair effects.

[0057] The fourth objective of this invention is to provide a method for improving the mechanical strength of collagen hydrogels while maintaining the light and dark streaks of collagen, by preparing collagen hydrogels using collagen single chains;

[0058] The collagen single chain has the following structure:

[0059] From the N-terminus to the C-terminus are: the first repeat sequence, the collagen domain, and the second repeat sequence;

[0060] Specifically, 2 to 5 cysteine ​​residues are introduced into the repeating sequences and / or collagen domains of collagen single chains;

[0061] The introduction is the insertion of cysteine, or the replacement of an amino acid with cysteine.

[0062] In one embodiment, 2 to 5 cysteine ​​residues are introduced into the repeating sequence and collagen domain of the collagen single chain.

[0063] In one embodiment, the collagen single chain has the following structure:

[0064] The sequence from the N-terminus to the C-terminus consists of a folded domain, a first repeat sequence, a collagen domain, and a second repeat sequence; wherein the amino acid sequence of the folded domain is shown in SEQ ID NO.1; the amino acid sequences of the first and second repeat sequences are shown in SEQ ID NO.2; and the amino acid sequence of the collagen domain is shown in SEQ ID NO.3.

[0065] Two to five cysteine ​​residues are introduced into the repeating sequences and collagen domains of collagen single chains. The introduction is either by inserting cysteine ​​residues or by replacing amino acids with cysteine ​​residues.

[0066] In one embodiment, one cysteine ​​residue is introduced at the beginning and end of the second repeat sequence, respectively;

[0067] Alternatively, one cysteine ​​residue may be introduced at the end of the first repeat sequence and the end of the second repeat sequence;

[0068] Optionally, 1 to 3 cysteine ​​residues are introduced into the collagen domain;

[0069] Optionally, one cysteine ​​residue is introduced into the collagen domain;

[0070] Optionally, two cysteine ​​residues are introduced into the collagen domain, with the two cysteine ​​residues separated by 40 to 80 amino acids;

[0071] Optionally, three cysteine ​​residues are introduced into the collagen domain, with the three cysteine ​​residues located at amino acids 1-40, 41-80, and 81-12 of the collagen domain, respectively.

[0072] A fifth object of the present invention is to provide the application of any of the above-mentioned collagen single chains or the above-mentioned genes, plasmids, cells or the above-mentioned collagen products in the fields of biology, food, chemical industry, medicine, biomaterials, tissue engineering or cosmetics.

[0073] This invention also provides a method for controlling the mechanical properties and morphology of hydrogels, wherein the method controls the expression of collagen molecules CL-domain and (PPG) in microbial cells. 10 The number of Cys residues in the amino acid sequence, as well as the CL-domain and (PPG) 10 The number of repetitions. Beneficial effects:

[0074] 1. This invention uses N-terminal and C-terminal (PPG) 10 Based on the sequence, a continuous Xaa-Yaa-Gly triplet collagen sequence is inserted in the middle, forming a three-segment chimeric collagen P-CL-P pattern. This is achieved through N-terminal and C-terminal (PPG)... 10 The three helices interact and self-assemble into type I collagen fibers with periodic light and dark stripes. This is achieved through the interaction of (PPG) 10 Introducing Cys into the neutralized CL-domain promotes further covalent cross-linking of collagen fibers to form a hydrogel.

[0075] 2. This invention utilizes cold shock expression of collagen sequences in *E. coli* to prepare clean collagen that can self-assemble into solutions and hydrogels with a type I collagen fiber structure. Its structure is similar to type I collagen fibers. The process is simple, low-cost, and suitable for large-scale production. This invention provides a preparation method and sequence design pattern for type I collagen fiber-like materials. The collagen region of this sequence is replaceable and expandable, providing a platform for the research and application of periodic collagen fibers based on light and dark stripes, and has broad prospects in biomaterials applications.

[0076] 3. This invention also regulates the mechanical properties and microstructure of the hydrogel by adjusting the amount of Cys in the collagen region. This is achieved by controlling (PPG) 10 The replacement location and number of Cys in collagen regions can control the cross-linking of type I collagen fibers to form hydrogels; by controlling the replacement number of Cys in CL-domain collagen regions, the mechanical properties and microstructure of the hydrogels formed by the cross-linking of type I collagen fibers can be controlled.

[0077] 4. The collagen prepared by this invention is non-cytotoxic and can promote the adhesion, extension and osteogenic differentiation of osteoblast precursor cells MC3T3-E1, as well as promote fracture repair in SD rats. Attached Figure Description

[0078] Figure 1 is a schematic diagram of the sequence design;

[0079] Figure 2 shows the SDS-PAGE identification of collagen;

[0080] Figure 3 shows the molecular weight determination of the designed collagen MALDI-TOF; a to f show the molecular weight determination of MALDI-TOF.

[0081] Figure 4 shows the determination of the secondary structure of the designed collagen; a is the full-wavelength scan spectrum of circular dichroism chromatography; b is the thermal curve of circular dichroism chromatography.

[0082] Figure 5 shows the self-assembled fiber morphology of mC, CC, and NC under natural, oxidizing, and reducing environments; where a, d, and g are the self-assembled fiber morphologies of mC under natural, oxidizing, and reducing environments, respectively; b, e, and h are the self-assembled fiber morphologies of CC under natural, oxidizing, and reducing environments, respectively; and c, f, and i are the self-assembled fiber morphologies of NC under natural, oxidizing, and reducing environments, respectively.

[0083] Figure 6 shows the assembled particle size and hydrogel mechanical properties of mC, CC, and NC; where a represents the particle size of mC, CC, and NC under reducing (DTT) and oxidizing (H2O2) conditions, and b represents the mechanical properties of hydrogels prepared from mC, CC, and NC.

[0084] Figure 7 shows the internal structures of mC, CC, and NC hydrogels; where a, d, and g are the internal structures of mC, b, e, and h are the internal structures of CC, and c, f, and i are the internal structures of NC.

[0085] Figure 8 shows the morphology of self-assembled fibers of NC1, NC3 and NC5 under natural, oxidative and reducing environments;

[0086] Figure 9 shows the assembled particle size and hydrogel mechanical properties of NC1, NC3 and NC5; where a is the particle size of NC1, NC3 and NC5 under reducing (DTT) conditions and oxidizing (H2O2) conditions; b is the mechanical properties of the hydrogels prepared from NC1, NC3 and NC5.

[0087] Figure 10 shows the internal structures of NC1, NC3, and NC5 hydrogels; where a and d are the internal structures of NC1 hydrogel; b and e are the internal structures of NC3 hydrogel; and c and f are the internal structures of NC5 hydrogel.

[0088] Figure 11 shows the biocompatibility and bioactivity verification of the designed type I collagen; where a is the cell proliferation results; b is the cell adhesion ability; c is the VCL mRNA expression level; d is the cell diffusion area; e is the cytoskeleton staining; f is the RUNX2 and Col1a1 mRNA expression levels; g is the ALP staining area; h is the ALP activity; and i is the ALP staining image of the entire well and the central region of the well.

[0089] Figure 12 shows the verification of the fracture repair function of NC1 collagen hydrogel; where a is micro-CT scan 4 weeks after repair; b is bone tissue parameter analysis; c is H&E and immunohistochemical staining analysis; and d is quantitative immunohistochemical staining analysis. Detailed Implementation

[0090] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0091] Some definitions or terms involved in this invention:

[0092] Type I collagen: Type I collagen is the only component of collagen fibers in the dermis, accounting for more than 80% of the skin; it is the most abundant component in dermal connective tissue. 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 very high tensile strength.

[0093] Type I collagen is the most abundant functional protein in animals. Its self-assembled collagen fibers, when characterized under a transmission electron microscope, exhibit alternating light and dark bands in overlapping and gap regions, commonly known as the D-cycle. The D-cycle is considered a key structural element conferring various functions of collagen and is related to the load-bearing properties of tissues, bone mineralization, and the regulation of cell differentiation and adhesion during tissue development.

[0094] Triple helix structure: Collagen is composed of three polypeptide chains forming a right-handed triple helix structure around a central axis. The triple helix structure can be further assembled to form higher-order collagen fibers, which perform their functions in the body. Therefore, the triple helix structure of collagen is the basis for its biological functions.

[0095] Collagen domain: also known as collagen structural domain or collagen domain, can be obtained by assembling and designing sequences from collagen fragments of human type I, II, and III collagen, or by assembling sequences from bacterial collagen.

[0096] In an optional embodiment, the collagen domain includes human collagen, bacterial collagen, recombinant human collagen, and recombinant bacterial collagen.

[0097] In an optional embodiment, the amino acid sequence of the collagen domain is shown in SEQ ID NO.3.

[0098] Folding domain: It helps collagen fold into a triple helix structure. Enzymatic digestion can remove the folding domain of the recombinant collagen, resulting in collagen that still retains the triple helix structure.

[0099] In an optional embodiment, the N-terminus of the first repeat sequence is connected to a folded domain, the amino acid sequence of which is shown in SEQ ID NO.1; in an optional embodiment, the folded domain is further provided with a 6×His tag at its front end.

[0100] In an optional embodiment, a linker peptide is present between the folded domain and the repeat sequence, the amino acid sequence of which is shown in LVPRGSPG (SEQ ID NO.4).

[0101] Repeating sequences: Repeating sequences can assist in the folding of the collagen triple helix and improve its thermal stability; there can be multiple repeating sequences, for example, located at both ends of the collagen domain or at both ends of multiple collagen domains, and named as the first repeating sequence and the second repeating sequence according to their position.

[0102] The repeating sequence modules are represented by (PPG)n. When there are multiple repeating sequence modules, the value of n in each repeating sequence module (PPG)n can be the same or different. By adjusting the number of n in the repeating sequence modules (PPG)n, the molecules can be further assembled to form a fibrous structure.

[0103] In an optional implementation, the first and second repeat sequences (PPG) n n is an integer from 4 to 30; preferably, n is from 5 to 10; in an optional embodiment, the amino acid sequences of the first and second repeating sequences are as shown in SEQ ID NO.2.

[0104] Collagen single chain: The collagen single chain includes the structure from the N-terminus to the C-terminus, the folding domain, the first repeat sequence, the collagen domain, and the second repeat sequence.

[0105] In an optional implementation, the structure of a collagen single chain is as follows:

[0106] Among them, the repeating sequence located at the N end (front end) of the CL-domain is the first repeating sequence, and the repeating sequence located at the C end (back end) of the CL-domain is the second repeating sequence.

[0107] Expression: As used in this article, “expression” refers to any step involving variant generation, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0108] Expression vector: As used herein, the term “expression vector” refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0109] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication, along with recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), and heterologous host cells.

[0110] Recombination: When used to refer to cells, nucleic acids, proteins, or vectors, the term "recombination" means that the cell has been modified from its natural state. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell, or express natural genes at different levels or under different conditions compared to those found in nature. The difference between recombinant nucleic acids and their natural sequences lies in the operative linking of one or more nucleotides and / or a foreign sequence (e.g., a foreign promoter in an expression vector). The difference between recombinant proteins and their natural sequences may lie in the fusion of one or more amino acids and / or a foreign sequence. A vector containing nucleic acids encoding a polypeptide is a recombinant vector. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0111] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0112] This invention provides a series of collagen single chains, wherein the collagen single chain has the following structure: from the N-terminus to the C-terminus: a first repeat sequence, a collagen domain, and a second repeat sequence; wherein 2 to 5 cysteine ​​residues are introduced into the repeat sequence and the collagen domain of the collagen single chain; the introduction is either by inserting cysteine ​​residues or by replacing amino acids with cysteine ​​residues.

[0113] Collagen single chains passing through the N-terminus and C-terminus (PPG) 10 The triple helices interact and self-assemble into type I collagen fibers with periodic light and dark stripes; by introducing cysteine ​​residues, the collagen fibers further covalently cross-link to form a hydrogel. The mechanical properties and microstructure of the hydrogel are modulated by adjusting the number of Cys residues in the collagen regions. This is achieved by controlling the amount of poly(p-glycosylated) collagen (PPG). 10 The replacement location and number of Cys in collagen regions can control the cross-linking of type I collagen fibers to form hydrogels; by controlling the replacement number of Cys in CL-domain collagen regions, the mechanical properties and microstructure of the hydrogels formed by the cross-linking of type I collagen fibers can be controlled.

[0114] In an optional embodiment, one cysteine ​​residue is introduced at the beginning and end of the second repeat sequence, respectively; or, one cysteine ​​residue is introduced at the end of the first repeat sequence and the second repeat sequence, respectively.

[0115] In an optional embodiment, based on the introduction of cysteine ​​into the repeating sequence, 1 to 3 cysteines are further introduced into the collagen domain; for example, the further introduction of 1 to 3 cysteines into the collagen domain includes: introducing 1 cysteine ​​into the collagen domain; or, introducing 2 cysteines into the collagen domain, with the 2 cysteines spaced 40 to 80 amino acids apart; or, introducing 3 cysteines into the collagen domain, with the 3 cysteines located at amino acids 1 to 40, 41 to 80, and 81 to 120, respectively, in the collagen domain.

[0116] Preferably, in addition to introducing cysteine ​​into the repeating sequence, one cysteine ​​is introduced into the collagen domain.

[0117] In an optional embodiment, the collagen single chain is as shown in any one of SEQ ID NO. 8 to SEQ ID NO. 11; the folded and enzyme-digested collagen single chain is as shown in any one of SEQ ID NO. 18 to SEQ ID NO. 21.

[0118] In an alternative embodiment, an amino acid is replaced with C at the end of the CL-domain, and a cysteine ​​residue is inserted at the end of the second repeat sequence.

[0119] In an optional embodiment, the Q (glutamine) at position 119 of the CL-domain (SEQ ID NO.3) is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, the amino acid sequence of which is shown in SEQ ID NO.8.

[0120] In an alternative implementation, an amino acid is replaced with C at the front end of the CL-domain, and a cysteine ​​residue is inserted at the end of the second repeat sequence.

[0121] In an optional embodiment, the first position (N-terminus) P of the CL-domain (SEQ ID NO.3) is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, the amino acid sequence of which is shown in SEQ ID NO.9.

[0122] In an optional embodiment, the first P in the CL-domain (SEQ ID NO.3) is replaced with C, and based on the insertion of a cysteine ​​residue at the end of the second repeat sequence (i.e., based on the collagen single chain with the amino acid sequence as shown in SEQ ID NO.9), the amino acid (X or Y in GXY) in the CL-domain is replaced with C.

[0123] In an optional embodiment, one X or Y is replaced with C in the first 1 / 3 of the peptide in the CL-domain (amino acids 1 to 40 of the CL-domain);

[0124] In an optional embodiment, one X or Y is replaced with C in the middle 1 / 3 peptide segment of the CL-domain (amino acids 41 to 80 of the CL-domain);

[0125] In an optional implementation, one X or Y is replaced with C in the last third of the peptide segment of the CL-domain (amino acids 81 to 120 of the CL-domain).

[0126] In an optional implementation, the first P in the CL-domain is replaced with C, and based on the insertion of a cysteine ​​residue at the end of the second repeat sequence, two X or Y residues in the CL-domain are replaced with C.

[0127] In an optional embodiment, one X or Y is replaced with C in the first half of the peptide in the CL-domain (amino acids 1 to 60 of the CL-domain), and one X or Y is replaced with C in the last half of the peptide in the CL-domain (amino acids 61 to 120 of the CL-domain).

[0128] In an optional implementation, the distance between the two replaced Cs is 40 to 80 amino acids.

[0129] In an optional implementation, the first P in the CL-domain is replaced with C, and a cysteine ​​residue is inserted at the end of the second repeat sequence, and three X or Y residues in the CL-domain are replaced with C.

[0130] In an optional embodiment, one X or Y is replaced with C in the first 1 / 3 of the peptide segment (amino acids 1 to 40 of the CL-domain), the middle 1 / 3 of the peptide segment (amino acids 41 to 80 of the CL-domain), and the last 1 / 3 of the peptide segment (amino acids 81 to 120 of the CL-domain), respectively.

[0131] In an optional implementation, the three replaced Cs are spaced 20 to 40 amino acids apart.

[0132] The present invention also provides a gene encoding any of the above-mentioned collagen single chains, a plasmid or cell carrying the gene encoding any of the above-mentioned collagen single chains;

[0133] In optional implementations, plasmids include the pColdIII series or the pET series.

[0134] In an optional embodiment, the cells are Escherichia coli cells, including but not limited to E. coli BL21 and E. coli BL21(DE3).

[0135] According to another aspect of the present invention, the present invention provides a method for preparing collagen hydrogel, comprising the steps of:

[0136] Collagen was prepared using the above method. The freeze-dried collagen was then prepared into a solution with a concentration of 0.1–1 mmol / L and allowed to stand at 4°C–37°C for at least 2 days to obtain type I collagen fibers.

[0137] After freeze-drying, the collagen is prepared into a solution with a concentration of ≥100g / L and left to stand at 4℃~37℃ for 1 week, or hydrogen peroxide is added and left to stand at room temperature for more than 2 hours to obtain type I collagen hydrogel.

[0138] In an optional implementation, type I collagen hydrogels exhibit excellent fracture repair effects.

[0139] According to another aspect of the present invention, the present invention provides a collagen product prepared from the above-described collagen single chains;

[0140] In an optional embodiment, the collagen product includes type I collagen, collagen fibers, and collagen hydrogel.

[0141] In an optional embodiment, the type I collagen is formed by the high-polymer self-assembly of the above-mentioned collagen single chains, and has a striped pattern of alternating light and dark.

[0142] In an optional embodiment, the collagen fibers are formed by the high-polymer self-assembly of the aforementioned type I collagen.

[0143] In optional embodiments, the collagen products include: collagen supplements, such as collagen peptides, collagen powder, collagen capsules / tablets, collagen liquids / oral solutions, etc.; collagen foods, such as collagen casings, collagen oatmeal, collagen gummies, functional drinks, etc.; medical devices and medical products, such as dressings / patches (for skin repair), injectable products (for filling wrinkles, improving skin texture, and repairing fractures), cold compresses / scar gels; beauty and skincare products, such as masks, serums / creams, sprays / repairing solutions; and biological raw materials and health products, such as active collagen raw materials (for cosmetics or biomedical materials).

[0144] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for illustrative purposes and should not be construed as limiting the present invention in any way. Preferred embodiments of the present invention will be described below. It should be understood that these embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0145] Materials and methods used in this invention:

[0146] Culture medium:

[0147] LB solid medium: 15 g / L agar, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0.

[0148] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0.

[0149] TB liquid medium: 12 g / L tryptone, 24 g yeast extract, 4 mL glycerol, 2.31 g KH2PO4, 12.54 g K2HPO4, pH 7.5, bring to a final volume of 1 L.

[0150] Cultivation methods:

[0151] Seed culture conditions: Single colonies grown from streaks on plates were inoculated into LB liquid medium with a medium volume of 10%, and cultured in 250mL shake flasks at a temperature of 37℃ for 10 hours at a rotation speed of 200rpm.

[0152] Fermentation culture conditions: TB medium was used with a medium volume of 20% and an inoculum size of 1%. The medium was cultured in 500 mL shake flasks at 37°C for 24 h. Then, IPTG at a final concentration of 1 mM was used for induction at 25°C for 10 h. The medium was then transferred to 15°C for 14 h of induction at a rotation speed of 200 rpm.

[0153] Example 1: Collagen Sequence Design and Sample Preparation

[0154] 1. Amino acid sequence design

[0155] The design follows the structure shown in VP-CL-P, where V stands for V-domain (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.16), representing the globular domain that guides the correct folding of the collagen region; P stands for (PPG). 10 , namely PPGPPGPPGPPGPPGPPGPPGPPGPPGPPG(SEQ ID NO.2); CL stands for CL-domain, i.e., collagen region.

[0156] Through (PPG) 10 The introduction of cysteine ​​(Cys) into the viscous module and CL-domain promotes further covalent cross-linking of collagen fibers to form a hydrogel (the introduction is the insertion of cysteine ​​or the replacement of amino acids with cysteine). A schematic diagram of the collagen sequence combination is shown in Figure 1.

[0157] The specific steps are as follows:

[0158] (1) The N and C ends of the sequence are (PPG) 10 The motif, with a CL-domain collagen region in the middle, yields a three-segment chimeric sequence (PPG). 10 -CL-domain-(PPG) 10 (abbreviated as P) 10 CLP 10 The structure is as follows:

[0159] Among them, CL-domain uses the collagen Scl2 (Genbank ID: AAL50184.1) derived from Streptococcus pyogenes to extract and splice amino acid sequences (amino acid sequences as shown in SEQ ID NO.3, nucleotide sequences as shown in SEQ ID NO.13), and inserts integrin binding sites (i.e., cysteine) in the middle of the collagen sequence to achieve its biological function.

[0160] (2) A globular domain V-domain (amino acid sequence as shown in SEQ ID NO.1) derived from Scl2 was inserted at the N-terminus of the sequence to guide the correct folding of the collagen triple helix. A linker peptide LVPRGSPG (SEQ ID NO.4) was inserted between the globular domain and the fixed sequence unit of the collagen region. 6×His was inserted at the N-terminus of the sequence for purification.

[0161] The amino acid sequence was constructed, and its name and specific amino acid sequence are shown in Table 1.

[0162] Table 1 Amino acid sequences

[0163] Among them, NC1, NC3, and NC5 are based on NC with an additional 3 to 5 Cs.

[0164] 2. Construction of recombinant strains

[0165] The pColdM plasmid was constructed by mutating the pColdIII plasmid using pColdM-S:CTCGAGGGATCCGAATTCA (SEQ ID NO.14) and pColdM-A:GAGCTCCATGGGCACTTTG (SEQ ID NO.15) as primers to introduce the Nco I site.

[0166] The genes corresponding to the amino acid sequences in Table 1 are synthesized, and their nucleotide sequences contain a 5' Nco I restriction site, a 5' flanking GC sequence, and a 3' Bam HI restriction site, respectively.

[0167] The synthesized genes were inserted between NcoI and BamHI in the pColdM plasmid to obtain the corresponding recombinant collagen protein plasmids. The recombinant plasmids were then transformed into E. coli BL21(DE3) competent cells using the CaCl2 method. The cells were plated on LB plates containing antibiotics, cultured, and screened to obtain recombinant strains for preparing heterozygous collagen. Based on the name of collagen, the recombinant strains were named E. coli-mC, E. coli-CC, E. coli-NC, E. coli-NC1, E. coli-NC3, and E. coli-NC5.

[0168] 3. Collagen Sample Preparation

[0169] After induction and fermentation of the recombinant strain, the fermentation broth was centrifuged at 8000 rpm for 5 minutes, and the bacterial cells were collected separately. The bacterial cells were resuspended in 10 mM phosphate buffer, and the cells were homogenized using an autoclave. Cell debris was then removed by centrifugation at 10000 rpm for 20 minutes at 4°C, and impurities were removed by filtration through a 0.45 μm microporous membrane. The sample was injected into a 5 mL His-Trap HP affinity chromatography column installed in a protein purification instrument. Eight column volumes were washed with washing buffer, and proteins were eluted with elution buffer containing an imidazole gradient (140 mM, 400 mM). The peak protein was collected for SDS-PAGE electrophoresis analysis. Then, the globular guide fold domain was removed by trypsin digestion at a final concentration of 0.05 mg / mL at 25°C for 8 hours. Following dialyzing to desalt, the collagen was freeze-dried to obtain lyophilized powder. Based on the sequence, the collagen was named mC, CC, NC, NC1, NC3, and NC5 (the amino acid sequences of the digested and folded collagen are shown in SEQ ID NO. 17–21). Small amounts of the lyophilized powder were dissolved in water and identified by SDS-PAGE and MALDI-TOF.

[0170] Figure 2 shows that the purified protein appears as a single band on SDS-PAGE. Since collagen is a rod-shaped protein, the molecular weight of the globular protein marker used on SDS-PAGE is larger than expected.

[0171] Figure 3 shows that the molecular weight obtained by mass spectrometry is consistent with the theoretical molecular weight, proving that the obtained collagen molecular weight is correct.

[0172] Example 2: Determination of collagen secondary structure

[0173] The collagen (mC, CC, NC, NC1, NC3, NC5) designed and prepared in Example 1 was prepared to a concentration of 1 mg / mL and incubated at 4°C for at least 24 hours. Circular dichroism spectroscopy was performed at 4°C using a 1 mm cuvette, scanning the entire wavelength range from 190 nm to 260 nm, with 1 nm intervals between wavelengths and a dwell time of 5 seconds at each wavelength. Thermochromatography was performed at 220 nm, with temperatures ranging from 4°C to 80°C, equilibrating at each temperature for 8 seconds, and a temperature increment rate of 1°C / 6 min. A typical triple-helix collagen CD spectrum showed a positive absorption peak at 225 nm.

[0174] As shown in Figure 4, under full-wavelength scanning, the collagen prepared in Example 1 has a characteristic absorption peak near 225 nm. The thermal distortion experiment results show that as the temperature increases, the characteristic absorption value at 225 nm changes abruptly between 37 and 55 °C, which is a manifestation of the destruction of the secondary structure of collagen, that is, the unwinding of the triple helix.

[0175] Both circular dichroism spectroscopy and thermal distortion tests showed that the collagen (mC, CC, NC, NC1, NC3, NC5) designed in Example 1 could fold correctly to form a triple helix structure of collagen and had high thermal stability.

[0176] Example 3: (PPG) 10 Effects of Cys substitution in different regions on fiber morphology

[0177] The freeze-dried collagen mC, CC, and NC prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mM using 10 mM PB, or 10 mM PB containing 0.1% H2O2 and 100 mM DTT was added. After being placed at 4°C for 3.5 days, a small amount was dropped onto a copper grid, adsorbed for 45 seconds, and then blotted dry with filter paper. The solution was then negatively stained with 0.75% phosphotungstic acid for 20 seconds, blotted dry with filter paper, and observed using a Hitachi H-7650 transmission electron microscope.

[0178] The transmission electron microscopy results are shown in Figure 5, in (PPG) 10 Introducing Cys into the motif affects the morphology of the self-assembled fiber. Under natural conditions, CC and NC form relatively good light and dark striped fibers. Under reducing conditions, mC, CC, and NC form obvious light and dark striped fibers because they are not affected by Cys crosslinking. Under oxidizing conditions, only NC can form good light and dark striped fibers.

[0179] Example 4: (PPG) 10 The Influence of Cys Replacement in Different Regions on Assembly, Hydrogel Mechanical Properties and Internal Structure

[0180] The collagen mC, CC, and NC obtained in Example 1 were tested for their properties, as detailed below:

[0181] 1. Collagen particle size detection

[0182] The lyophilized collagen mC, CC, and NC prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mg / mL. The buffer solution was 10 mM PB containing 0.1% H2O2 or 100 mM DTT. After being placed at 4°C for 3.5 days, the hydrated particle size of the assembled particles was determined by dynamic light scattering. As shown in Figure 6a, mC, CC, and NC all aggregated into large particles with a hydrated radius (Rh) of approximately 1000 nm. The particle size under oxidative conditions was larger than that under reducing conditions, with the NC aggregate having the largest particle size of 1828 nm.

[0183] 2. Collagen hydrogel performance testing

[0184] (1) Appearance and modulus of hydrogel

[0185] The lyophilized collagen mC, CC and NC prepared in Example 1 were prepared into a solution with a final concentration of 100 g / L, placed at 4°C for 3.5 days, and then 0.1% H2O2 was added for crosslinking at room temperature for 2 hours.

[0186] The rheological properties of the hydrogels are shown in Figure 6b. At a concentration of 10% w / v, mC, CC, and NC all exhibit hydrogels, with storage modulus G' > loss modulus G'. The modulus of NC is larger than that of mC and CC. The appearance of the hydrogels is shown in Figures 7a, b, and c. mC, CC, and NC form a translucent hydrogel. The sample does not flow in an inverted transparent centrifuge tube, forming a collagen hydrogel.

[0187] (2) Scanning electron microscopy of hydrogels

[0188] After freezing the small amount of hydrogel prepared in step (1) with liquid nitrogen, the sample was freeze-dried in a freeze dryer. After sputtering gold on the cross-section, the internal structure of the collagen hydrogel was observed using a scanning electron microscope. The results are shown in d, e, f, g, h, and i in Figure 7. The hydrogel samples of mC, CC, and NC all showed loose and porous collagen sponges. However, the internal structure of mC was an isotropic network microporous structure, while the internal structures of CC and NC were anisotropic layered stacked structures.

[0189] Example 5: Effect of different amounts of Cys substitution in the CL-domain region on fiber morphology

[0190] The freeze-dried collagen NC1, NC3, and NC5 prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mM using 10 mM PB, or 10 mM PB containing 0.1% H2O2 and 100 mM DTT was added. After being placed at 4°C for 3.5 days, a small amount was dropped onto a copper grid, adsorbed for 45 seconds, and then blotted dry with filter paper. The solution was then negatively stained with 0.75% phosphotungstic acid for 20 seconds, blotted dry with filter paper, and observed using a Hitachi H-7650 transmission electron microscope.

[0191] The transmission electron microscopy results are shown in Figure 8. Replacing different amounts of Cys in the CL-domain region affects the morphology of the self-assembled fibers; the more Cys present, the more severe the fiber damage. Under natural conditions, NC1 and NC3 can form fibers with light and dark stripes. Under reducing conditions, NC1 and NC3 form fibers with obvious light and dark stripes, while NC5 still cannot form fibers with light and dark stripes. Under oxidizing conditions, only NC1 can form fibers with relatively good light and dark stripes.

[0192] Example 6: Effects of different amounts of adhesive used in CL-domain region replacement on mechanical properties and internal structure

[0193] The collagen NC1, NC3, and NC5 obtained in Example 1 were tested for their properties, as detailed below:

[0194] 1. Collagen particle size detection

[0195] The lyophilized collagen NC1, NC3 and NC5 prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mg / mL. The buffer solution was 10 mM PB containing 0.1% H2O2 or 100 mM DTT. After being placed at 4°C for 3.5 days, the hydrated particle size of the assembled particles was determined by dynamic light scattering.

[0196] As shown in Figure 9a, NC1, NC3 and NC5 all aggregated into large particles. NC1 had the largest hydrated particle size (Rh) of 1427 nm, while NC5 had the smallest hydrated particle size of 375 nm. The particle size under oxidizing conditions was larger than that under reducing conditions, and the aggregate formed by NC1 had the largest particle size of 3580 nm.

[0197] 2. Collagen hydrogel performance testing

[0198] (1) Appearance and modulus of hydrogel

[0199] The freeze-dried collagen NC1, NC3 and NC5 prepared in Example 1 were prepared into a solution with a final concentration of 100 g / L, placed at 4°C for 3.5 days, and then 0.1% H2O2 was added for cross-linking at room temperature for 2 hours.

[0200] The rheological properties of the hydrogels are shown in Figure 9b. At a concentration of 10%, NC1, NC3, and NC5 all exhibit hydrogel characteristics. The storage modulus G' > loss modulus G'. The more Cys replaced in the CL-domain region, the weaker the mechanical properties of the hydrogel. As the number of Cys increases, the modulus of the hydrogel decreases, and the angular frequency reaching the sol point decreases. The appearance of the hydrogels is shown in Figures 10a, b, and c. NC1, NC3, and NC5 form translucent hydrogels. The samples do not flow in inverted transparent centrifuge tubes, forming collagen hydrogels. After gelation at 4°C for more than 2 weeks, NC1 can be picked up with pointed tweezers and has strong mechanical properties, while NC3 and NC5 cannot be picked up with pointed tweezers.

[0201] (2) Scanning electron microscopy of hydrogels

[0202] A small amount of hydrogel was frozen in liquid nitrogen and then freeze-dried in a freeze dryer. After sputtering gold onto the cross-section, the internal structure of the collagen hydrogel was observed using a scanning electron microscope, as shown in d, e, and f in Figure 10. The hydrogel samples of NC1, NC3, and NC5 all showed loose and porous collagen sponges. The internal structure of NC5 was an isotropic network-like microporous structure, while the internal structures of NC1 and NC3 were anisotropic layered stacked structures.

[0203] Example 7: Validation of the biocompatibility and bioactivity of the designed type I collagen

[0204] The lyophilized collagen mC, CC, NC, NC1, NC3 and NC5 prepared in Example 1 were prepared into a collagen solution with a final concentration of 40 ng / μL. Bovine serum albumin (BSA) and rat tail type I collagen at the same concentration were used as negative and positive controls, respectively.

[0205] 100 μL of collagen solution was added to each well of a 96-well plate, with three replicates per group. After adsorption at 4°C for 24 h, the solution was aspirated, dried overnight, and then sterilized by UV irradiation for 30 min. Finally, the plates were blocked with 5% BSA for 2 h. MC-3T3-E1 cells were resuspended in DMEM containing 10% FBS at a density of 2000 cells per well, and 100 μL was seeded onto cell culture plates. Cell viability was measured using a CCK8 assay kit at days 0, 1, 3, and 5.

[0206] As shown in Figure 11a, the designed collagen had a significant effect on cell proliferation without cytotoxicity. Collagen was adsorbed onto 96-well plates in the same manner, and 10,000 cells were seeded and cultured for 6 hours. After washing three times with 10 mM PBS, cell viability was measured using the CCK8 assay kit, as shown in Figures 11b and 11c. Compared to samples with poor fiber morphology, NC1 showed a significant promoting effect on cell adhesion, superior to natural Type I collagen. The expression level of Vinculin-related VCL mRNA was also significantly increased. As shown in Figures 11d-e, when cell diffusion area was assessed using the same method and cytoskeleton staining was performed with phalloidin, cells adhering to the NC1 matrix showed a larger diffusion area and more pronounced cytoskeleton extension, comparable to Type I. These results indicate that the synthesized collagen NC1 has excellent biocompatibility.

[0207] Furthermore, we verified the osteogenic differentiation capacity of MC3T3-E1 osteoblast precursor cells on the surface of designed collagen fibers. One week after inducing osteogenic differentiation in MC3T3-E1 cells, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining. As shown in Figures 11f-i, the ALP staining area and ALP activity of NC and NC1 were comparable to or even greater than those of type I collagen, significantly exceeding those of the mC, CC, NC3, NC5, or BSA sample groups.

[0208] Meanwhile, type I collagen fibers NC and NC1 induced the expression of osteoblast-specific transcription factor RUNX2 and the type I collagen α1 chain Col1a1 of the bone matrix protein gene, with levels exceeding those of other synthetic collagens and even higher than those of natural type I collagen. The review demonstrates that NC and NC1 collagen can promote osteogenic differentiation of MC3T3-E1 osteoblast precursor cells.

[0209] Example 8: Verification of the fracture repair function of NC1 collagen hydrogel

[0210] The lyophilized collagen NC1 prepared in Example 1 was prepared into a solution with a final concentration of 100 g / L and placed at 4°C for one week to form NC1 collagen hydrogel, thus verifying its fracture repair function.

[0211] Six-week-old male SD rats underwent a 1.5–2 cm incision on the lateral side of the femur. The mucosa and muscle were bluntly dissected to expose the midshaft of the femur. Intramedullary fixation was performed using 1.2 mm Kirschner wires, and the fracture ends were secured with 3-0 absorbable sutures. Postoperatively, the wound was thoroughly rinsed with 0.9% sodium chloride solution, and the muscle and skin were sutured. Penicillin was administered intramuscularly at 100,000 units per rat for 3 consecutive days postoperatively to prevent infection.

[0212] 200 μL of each of the following treatments were injected into the fracture area: blank control group (physiological saline), NC1 collagen hydrogel, and positive control group (rat tail type I collagen hydrogel). The injections were administered every other day for two weeks. Four weeks later, the rats were sacrificed, and micro-CT scans were performed to observe fracture repair. The remaining samples were decalcified and subjected to tissue sectioning, H&E staining, and immunohistochemical staining (VEGF, RUNX2, and ALP) to observe angiogenesis and the expression levels of bone synthesis-related proteins.

[0213] The results are shown in Figure 12. Micro-CT results showed that, compared with the untreated drug-treated model, NC1 collagen hydrogel exhibited a significant fracture repair effect, which was very close to that of natural type I hydrogel from rat tail. Bone-related parameter analysis also showed that the fracture area treated with NC1 collagen hydrogel had significantly increased bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number, and connective tissue density.

[0214] H&E staining results showed no obvious inflammatory cell infiltration, and there was no significant difference between the groups. Immunohistochemical staining results are shown in Figure 12c. The vascular endothelial growth factor (VEGF) in the fracture area of ​​SD rats treated with NC1 collagen hydrogel was significantly increased, indicating that angiogenesis was active in the local microenvironment of the fracture, and that NC1 collagen hydrogel has the ability to promote angiogenesis.

[0215] Compared with the control group, the protein expression level and alkaline phosphatase (ALP) activity of Runx2, a core transcription factor for osteogenic differentiation, were significantly increased in the NC1 collagen hydrogel group (p<0.0005), and were higher than those in rat tail collagen type I hydrogel.

[0216] In summary, the results indicate that NC1 collagen hydrogel can significantly promote the osteogenic phenotype transformation of mesenchymal stem cells by upregulating the Runx2-mediated osteogenic differentiation pathway, and has a significant promoting effect on fracture repair.

[0217] The amino acid sequence used in this invention is as follows:

[0218] The amino acid sequence of the V-domain (SEQ ID NO.1):

[0219] The amino acid sequence of the CL-domain (SEQ ID NO.3):

[0220] Nucleotide sequence of the CL-domain (SEQ ID NO.13)

[0221] The nucleotide sequence of the V-domain (SEQ ID NO.16)

[0222] Folded and enzyme-digested mC (SEQ ID NO.17)

[0223] Folded and enzyme-digested CC (SEQ ID NO.18)

[0224] Folded and enzyme-digested NC (SEQ ID NO.19)

[0225] Folded and enzyme-digested NC1 (SEQ ID NO.20)

[0226] Folded and enzyme-digested NC3 (SEQ ID NO.21)

[0227] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A collagen single chain, characterized in that, The collagen single chain has the following structure: From the N-terminus to the C-terminus are: the first repeat sequence, the collagen domain, and the second repeat sequence; Among them, 2 to 5 cysteine ​​residues are introduced into the repeat sequences and collagen domains of collagen single chains; The introduction is the insertion of cysteine, or the replacement of an amino acid with cysteine.

2. The collagen single chain according to claim 1, characterized in that, One cysteine ​​residue is introduced at the beginning and one at the end of the second repeat sequence; Alternatively, one cysteine ​​residue may be introduced at the end of the first repeat sequence and the end of the second repeat sequence.

3. The collagen single chain according to claim 2, characterized in that, Introduce 1 to 3 cysteine ​​residues into the collagen domain.

4. The collagen single chain according to claim 3, characterized in that, One cysteine ​​residue is introduced into the collagen domain.

5. The collagen single chain according to claim 3, characterized in that, Two cysteine ​​residues are introduced into the collagen domain, with a spacing of 40 to 80 amino acids between them.

6. The collagen single chain according to claim 3, characterized in that, Three cysteine ​​residues were introduced into the collagen domain. These three cysteine ​​residues were located in amino acids 1–40, 41–80, and 81–120 of the collagen domain, respectively.

7. The collagen single chain according to any one of claims 1 to 6, characterized in that, A folded domain is attached to the N-terminus of the first repeat sequence, and the amino acid sequence of the folded domain is shown in SEQ ID NO.

1.

8. The collagen single chain according to any one of claims 1 to 6, characterized in that, The amino acid sequences of the first and second repeat sequences are (PPG). n n is an integer between 4 and 30.

9. The collagen single chain according to any one of claims 1 to 6, characterized in that, The collagen domain includes human collagen, bacterial collagen, recombinant human collagen, and recombinant bacterial collagen.

10. The collagen single chain according to any one of claims 1 to 6, characterized in that, The amino acid sequence of the collagen domain is shown in SEQ ID NO.

3.

11. The collagen single chain according to any one of claims 1 to 6, characterized in that, There is a linker peptide between the folded domain and the repeating sequence, the amino acid sequence of which is shown in SEQ ID NO.

4.

12. The collagen single chain according to any one of claims 1 to 6, characterized in that, The amino acid sequence of the collagen single chain is shown in any one of SEQ ID NO. 8 to SEQ ID NO. 11; the amino acid sequence of the collagen single chain after enzymatic digestion and folding is shown in any one of SEQ ID NO. 18 to SEQ ID NO.

21.

13. A gene encoding a single strand of collagen as described in any one of claims 1 to 12, a plasmid or cell carrying a gene encoding a single strand of collagen as described in any one of claims 1 to 11; in, Plasmids include the pColdIII series or the pET series.

14. The collagen product obtained by preparing collagen single chains according to any one of claims 1 to 12.

15. The collagen product according to claim 14, characterized in that, The collagen products include type I collagen, collagen fibers, and collagen hydrogels.

16. A method for improving the mechanical strength of collagen hydrogel while maintaining the light and dark streaks of collagen, characterized in that, Collagen hydrogels were prepared using single-chain collagen. From the N-terminus to the C-terminus are: the first repeat sequence, the collagen domain, and the second repeat sequence; Among them, 2 to 5 cysteine ​​residues are introduced into the repeat sequences and collagen domains of collagen single chains; The introduction is the insertion of cysteine, or the replacement of an amino acid with cysteine.

17. The method according to claim 16, characterized in that, One cysteine ​​residue is introduced at the beginning and one at the end of the second repeat sequence; Alternatively, one cysteine ​​residue may be introduced at the end of the first repeat sequence and the end of the second repeat sequence.

18. The method according to claim 17, characterized in that, Introduce 1–3 cysteine ​​residues into the collagen domain; One cysteine ​​residue was introduced into the collagen domain; Alternatively, two cysteine ​​residues can be introduced into the collagen domain, with the two cysteine ​​residues separated by 40 to 80 amino acids; Alternatively, three cysteine ​​residues can be introduced into the collagen domain, located at amino acids 1-40, 41-80, and 81-12, respectively.

19. The method according to claim 17, characterized in that, The method described in any one of 16 to 18 is characterized in that a folding domain is attached to the N-terminus of the first repeating sequence, and the amino acid sequence of the folding domain is shown in SEQ ID NO.1; The amino acid sequences of the first and second repeat sequences are (PPG). n n is an integer from 4 to 30; The collagen domain includes human collagen, bacterial collagen, recombinant human collagen, and recombinant bacterial collagen; the amino acid sequence of the collagen domain is shown in SEQ ID NO.3; There is a linker peptide between the folded domain and the repeating sequence, the amino acid sequence of which is shown in SEQ ID NO.

4.

20. The use of the collagen single chain of any one of claims 1 to 12, or the gene, plasmid, cell of claim 6, or the collagen product of claim 14, or the method of any one of claims 16 to 19, in the fields of biology, food, chemical industry, medicine, biomaterials, tissue engineering, or cosmetics.

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