Biosynthetic I-type collagen hydrogel for fracture repair and preparation method of biosynthetic I-type collagen hydrogel

Abstract

The invention discloses a biosynthetic type I collagen hydrogel for fracture repair and a preparation method thereof, and belongs to the technical field of genetic engineering. PPG 10 is expressed at the N end and the C end of collagen in a fusion mode, high polymerization self-assembly of the collagen is promoted to form I-type collagenous fiber collagen fibers with periodical alternately dark and bright stripes, and Cys is introduced into CL-domain in the PPG 10 in an oriented design mode to promote further covalent crosslinking of the collagen fibers to form the hydrogel. The collagen fiber structure in the hydrogel prepared by the invention is similar to that of natural I-type collagen fiber, the hydrogel has good biocompatibility and bone repair function, the preparation process is simple, the I-type collagen fiber hydrogel with low cost can be produced on a large scale, and the hydrogel has a wide application prospect in the field of biological materials.

Description

Technical Field

[0001] The present invention relates to a biosynthetic type I collagen hydrogel for fracture repair and a preparation method thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Collagen, composed of three polypeptide chains entwined in 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, regulating cell proliferation and differentiation and playing a key role in the repair and regeneration of tissues and organs. There are 28 types of collagen in mammals, with type I being the most common. In the cells of higher organisms, type I collagen undergoes translation, modification, folding, and cleavage to form a regularly arranged, ribbon-like fibrous structure. This ribbon-like structure, characterized by alternating dark and light bands, is known to be crucial 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 fibrils exhibit a pattern of alternating light and dark stripes. This unique periodic pattern in collagen is formed by the offset and parallel assembly of the collagen triple helix, which is crucial for cell activity, osteoblast differentiation, and matrix mineralization.

[0003] Type I collagen, extracted from animals, has a wide range of applications as a biomaterial in food and cosmetics, drug delivery, and biomedical engineering. It can be used in cell culture, tissue engineering, and regenerative medicine. It can be used as a scaffold or matrix material in biomedical materials such as skin burn dressings, hemostatic sponges for surgical and dental procedures, and bone defect filling to promote tissue regeneration and repair, generating significant market demand. Currently, type I collagen on the market is primarily derived from connective tissues such as animal skin and Achilles tendons. While it boasts high biocompatibility and ease of absorption, it is susceptible to contamination by animal-derived diseases, such as prions. To improve biosafety, the preparation of cleanly sourced type I collagen is a key issue in the field of biomedical materials.

[0004] Currently, there are four main sources of collagen: 1. Animal extraction: low price, but easy to carry pathogens; 2. Chemical synthesis: high purity and strong controllability, but expensive, limited in length, and not suitable for mass production; 3. Expression in eukaryotic systems such as transgenic plants and mammalian cells: can correctly fold proteins and perform complex post-translational processing, but has high culture costs, long cycles, low expression levels, and is difficult to mass produce; 4. Microbial expression system: has obvious advantages such as low cost and high expression levels.

[0005] Research has 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 a collagen triple helix structure, demonstrating the advantages of low cost and high expression yield. However, most recombinant collagens still lack the driving force for self-assembly, unable to polymerize into hydrogels with periodic striations or specific structures similar to native type I collagen fibrils. This limits their inherent advantages in cell viability, osteogenic differentiation, and biomineralization, hindering their potential for application in biomaterials and tissue engineering.

[0006] Collagen hydrogels are biodegradable, water-rich, three-dimensional matrices that can provide biological signals and act as a scaffold for cells, creating the necessary microenvironment for cell growth, differentiation, and migration, making them suitable for bone repair. Therefore, the preparation of collagen hydrogels with distinct light and dark stripes and excellent physiological activity has extremely high practical and economic value. Summary of the Invention

[0007] This application is expressed by fusing the N-terminus and C-terminus of collagen from different sources (PPG) 10 , promotes the self-assembly of collagen to form collagen fibers, and through (PPG) 10 The introduction of Cys into the neutralized CL-domain promoted 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 structure:

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

[0010] wherein 2 to 5 cysteines are introduced into the repeat sequence and / or collagen domain of the collagen single chain;

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

[0012] Optionally, 2 to 5 cysteines are introduced into the repeat sequence and collagen domain of the collagen single chain.

[0013] In one embodiment, a cysteine (designated CC) is introduced at the front and end of the second repeat sequence respectively;

[0014] Alternatively, a cysteine (designated as NC) is introduced at the end of each of the first and second repeat sequences.

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

[0016] Optionally, a cysteine is introduced into the collagen domain;

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

[0018] Optionally, three cysteines are introduced into the collagen domain, and the three cysteines are located in amino acids 1 to 40, 41 to 80, and 81 to 120 of the collagen domain, respectively.

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

[0020] From N-terminus to C-terminus: the first repeat sequence (PPG) n , collagen domain (CL-domain), second repeat sequence (PPG) n.

[0021] In one embodiment, a folding domain is connected 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 end of the folding domain also includes a 6×His tag.

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

[0024] There is a connecting peptide between the folded domain and the repeat sequence, the amino acid sequence of which is shown as 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 5 to 10; preferably, the amino acid sequence of the first and second repeating sequences is as shown in SEQ ID NO.2;

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

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

[0028] In one embodiment, in the structure of a single collagen chain, the repeating sequence located at the N-terminus (front end) of the CL-domain is the first repeating sequence, and the repeating sequence located at the C-terminus (back end) of the CL-domain is the second repeating sequence.

[0029] In one embodiment, the collagen single chain is shown in any one of SEQ ID NO.8 to SEQ ID NO.11; the collagen single chain after folding and enzyme cleavage is shown in any one of SEQ ID NO.18 to SEQ ID NO.21.

[0030] In one embodiment, an amino acid is substituted with C at the end of the CL-domain and a cysteine is inserted at the end of the second repeat sequence;

[0031] Optionally, the Q (glutamine) at position 119 of the CL-domain (SEQ ID NO.3) is replaced by C, and a cysteine 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.8.

[0032] In one embodiment, an amino acid is substituted with C at the front end of the CL-domain, and a cysteine is inserted at the end of the second repeat sequence;

[0033] Optionally, the first position (N-terminus) P of CL-domain (SEQ ID NO.3) is replaced by C, and a cysteine 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.

[0034] In one embodiment, the first P in the CL-domain (SEQ ID NO. 3) is replaced with a C, and a cysteine is inserted at the end of the second repeat sequence (i.e., based on the amino acid sequence of the collagen single chain shown in SEQ ID NO. 9), the amino acid (X or Y in GXY) in the CL-domain is replaced with a C;

[0035] Alternatively, 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 by C;

[0036] Alternatively, 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 a C;

[0037] Alternatively, one X or Y is replaced by C in the last 1 / 3 of the CL-domain peptide (amino acids 81 to 120 of the CL-domain).

[0038] In one embodiment, the first P in the CL-domain is replaced by a C, and a cysteine is inserted at the end of the second repeat sequence, and two X or Y in the CL-domain are replaced by C;

[0039] Alternatively, in the first 1 / 2 of the peptide in the CL-domain (amino acids 1 to 60 of the CL-domain), one X or Y is replaced by C, and in the second 1 / 2 of the peptide in the CL-domain (amino acids 61 to 120 of the CL-domain), one X or Y is replaced by C;

[0040] Optionally, the two substituted Cs are 40 to 80 amino acids apart.

[0041] In one embodiment, the first P in the CL-domain is replaced by a C, and a cysteine is inserted at the end of the second repeat sequence, and three X or Y in the CL-domain are replaced by C;

[0042] Alternatively, in the first 1 / 3 peptide segment (amino acids 1 to 40 of the CL-domain), the middle 1 / 3 peptide segment (amino acids 41 to 80 of the CL-domain), and the last 1 / 3 peptide segment (amino acids 81 to 120 of the CL-domain), one X or Y is replaced by C, respectively;

[0043] Optionally, the three substituted Cs are spaced 20 to 40 amino acids apart from each other.

[0044] The second object of the present invention is to provide a gene encoding any of the above-mentioned collagen single chains, a plasmid or a cell carrying a gene encoding any of the above-mentioned collagen single chains;

[0045] Optionally, the plasmid includes the pColdIII series or the pET series.

[0046] In one embodiment, the cell is an Escherichia coli cell, including but not limited to E. coli BL21 and E. coli BL21 (DE3).

[0047] The third object of the present invention is to provide a collagen product prepared from any of the above-mentioned collagen single chains;

[0048] Optionally, the collagen product includes type I collagen, collagen fibers and collagen hydrogel.

[0049] In one embodiment, the type I collagen is formed by the self-assembly of the above-mentioned collagen single chains and has a light and dark stripe morphology.

[0050] In one embodiment, the collagen fibers are formed by self-assembly of the above-mentioned type I collagen.

[0051] In one embodiment, the preparation method of the collagen hydrogel is:

[0052] Purify, dialyze, and freeze-dry the collagen produced by fermenting the above-mentioned collagen gene, a plasmid carrying a gene encoding any of the above-mentioned collagen proteins, or a cell;

[0053] The lyophilized collagen is prepared into a solution with a concentration of 0.1 to 1 mmol / L, and allowed to stand at 4°C to 37°C for at least 2 days to obtain type I collagen fibers;

[0054] The freeze-dried collagen is prepared into a solution with a concentration greater than or equal to 100 g / L, and is allowed to stand at 4°C to 37°C for 1 week, or hydrogen peroxide is added and allowed to stand at room temperature for more than 2 hours to obtain a type I collagen hydrogel.

[0055] In one embodiment, the type I collagen hydrogel has excellent fracture repair effect.

[0056] The fourth object of the present invention is to provide a method for improving the mechanical strength of collagen hydrogel while maintaining the light and dark stripes of collagen, and to prepare collagen hydrogel using single collagen chains;

[0057] The collagen single chain has the structure:

[0058] From N-terminus to C-terminus: first repeat sequence, collagen domain, second repeat sequence;

[0059] wherein 2 to 5 cysteines are introduced into the repeat sequence and / or collagen domain of the collagen single chain;

[0060] The introduction is the insertion of cysteine or the substitution of an amino acid with cysteine.

[0061] In one embodiment, a total of 2 to 5 cysteines are introduced into the repeat sequence and collagen domain of the collagen single chain.

[0062] In one embodiment, the collagen single chain has the structure:

[0063] From the N-terminus to the C-terminus are: the folding domain, the first repeat sequence, the collagen domain, and the second repeat sequence; wherein 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 shown in SEQ ID NO.2; and the amino acid sequence of the collagen domain is shown in SEQ ID NO.3;

[0064] 2 to 5 cysteines are introduced into the repeating sequence and collagen domain of the collagen single chain, and the introduction is by inserting cysteine or replacing amino acids with cysteine.

[0065] In one embodiment, one cysteine is introduced at the front end and the end of the second repeat sequence respectively;

[0066] Alternatively, one cysteine is introduced at each end of the first repeat sequence and the second repeat sequence;

[0067] Optionally, 1 to 3 cysteines are introduced into the collagen domain;

[0068] Optionally, a cysteine is introduced into the collagen domain;

[0069] Optionally, two cysteines are introduced into the collagen domain, with the two cysteines separated by 40 to 80 amino acids;

[0070] Optionally, three cysteines are introduced into the collagen domain, and the three cysteines are located in amino acids 1 to 40, 41 to 80, and 81 to 12 of the collagen domain, respectively.

[0071] A fifth object of the present invention is to provide applications 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 engineering, medicine, biomaterials, tissue engineering or cosmetics.

[0072] The present invention also provides a method for controlling the mechanical properties and morphology of hydrogels, wherein the method is to control the collagen molecules CL-domain and (PPG) expressed by microbial cells. 10 The number of Cys residues in the amino acid sequence and the relationship between CL-domain and (PPG) 10 The number of repetitions.

[0073] Beneficial effects:

[0074] 1. The present invention uses N and C termini (PPG) 10 The collagen sequence of continuous Xaa-Yaa-Gly triplets is inserted in the middle to form a three-segment chimeric collagen P-CL-P pattern. 10 The triple helices interact with each other and self-assemble into type I collagen fibers with periodic light and dark stripes. 10 The introduction of Cys into the neutralized CL-domain promoted further covalent cross-linking of collagen fibers to form a hydrogel.

[0075] 2. This invention utilizes cold-shock expression of collagen sequences in Escherichia coli to produce clean collagen that self-assembles into solutions and hydrogels with type I collagen fibrils. This structure is similar to type I collagen fibers, resulting in a simple process, low cost, and amenable to large-scale production. This invention provides a preparation method and sequence design for the production of type I collagen-like fibers. The collagen regions of this sequence are interchangeable and scalable, providing a platform for the research and application of periodic light-dark striped collagen fibers, and holds broad promise for biomaterial applications.

[0076] 3. The present invention also regulates the mechanical properties and microstructure of the hydrogel by adjusting the amount of Cys in the collagen region. By controlling (PPG) 10 The replacement position and number of Cys in the collagen region can control the cross-linking of type I collagen fibers to form a hydrogel; by controlling the number of Cys replacements in the CL-domain collagen region, the mechanical properties and microstructure of the cross-linked hydrogel formed by type I collagen fibers can be controlled.

[0077] 4. The collagen prepared by the present 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Design schematics for sequences;

[0079] Figure 2 For collagen SDS-PAGE identification;

[0080] Figure 3 is the designed collagen MALDI-TOF molecular weight identification; a~f are MALDI-TOF molecular weight identification.

[0081] Figure 4 The secondary structure of the designed collagen is determined; a is the full wavelength scanning spectrum of the circular dichroism spectrum; b is the thermal curve of the circular dichroism spectrum.

[0082] Figure 5 The self-assembled fiber morphologies of mC, CC and NC under natural, oxidative and reductive environments; a, d, g are the self-assembled fiber morphologies of mC under natural, oxidative and reductive environments, respectively; b, e, h are the self-assembled fiber morphologies of CC under natural, oxidative and reductive environments, respectively; c, f, i are the self-assembled fiber morphologies of NC under natural, oxidative and reductive environments, respectively;

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

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

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

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

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

[0088] Figure 11 Verification of the biocompatibility and bioactivity of the designed type I collagen; where a is the cell proliferation result; b is the cell adhesion ability; c is the VCL mRNA expression level; d is the cell spreading 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; i is the ALP staining image of the entire well and the center area of the well;

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

[0090] Materials and methods used in the present invention:

[0091] Culture medium:

[0092] LB solid medium: 15 g / L agar, 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L NaCl, pH 7.0.

[0093] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L NaCl, pH 7.0.

[0094] TB liquid medium: 12 g / L tryptone, 24 g yeast extract powder, 4 mL glycerol, 2.31 g KH2PO4, 12.54 g K2HPO4, pH 7.5, adjust the volume to 1 L.

[0095] Cultivation method:

[0096] Seed culture conditions: a single colony grown from a streak on a plate was inoculated into LB liquid medium with a medium volume of 10%. Culture was performed in a 250 mL shake flask at 37°C for 10 h at a rotation speed of 200 rpm.

[0097] Fermentation culture conditions: TB medium was used with a medium filling volume of 20% and an inoculum size of 1%. Culture was performed in a 500 mL shake flask. After culturing at 37°C for 24 h, IPTG was induced with a final concentration of 1 mM. The culture was induced at 25°C for 10 h, and then at 15°C for 14 h at a rotation speed of 200 rpm.

[0098] Example 1: Collagen sequence design and sample preparation

[0099] 1. Amino acid sequence design

[0100] The design was based on the structure shown in VP-CL-P, where V is the V-domain (amino acid sequence shown in SEQ ID NO.1, nucleotide sequence shown in SEQ ID NO.16), representing the globular domain that guides the correct folding of the collagen region; P is (PPG) 10 , i.e. PPGPPGPPGPPGPPGPPGGPPGGPPGGPPG (SEQ ID NO.2); CL represents CL-domain, i.e. collagen region.

[0101] Through (PPG) 10 The introduction of cysteine (Cys) into the CL-domain (i.e., the adhesive module) 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). The schematic diagram of the collagen sequence combination is shown in FIG. Figure 1 shown.

[0102] The specific steps are as follows:

[0103] (1) The N and C termini of the sequence are (PPG) 10 Motif, with the CL-domain collagen region in the middle, resulting in a three-segment chimeric sequence (PPG) 10-CL-domain-(PPG) 10 (abbreviated as P 10 CLP 10 ), wherein the CL-domain adopts the amino acid sequence of collagen Scl2 (Genbank ID: AAL50184.1) derived from Streptococcus pyogenes (the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.13), and an integrin binding site (i.e., cysteine) is inserted in the middle of the collagen sequence to achieve its biological function;

[0104] (2) The globular domain V-domain (amino acid sequence 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. The connecting peptide LVPRGSPG (SEQ ID NO. 4) was inserted between the fixed sequence unit of the globular domain and the collagen region. 6×His was inserted at the N-terminus of the sequence for purification.

[0105] The amino acid sequences were constructed, and the names and amino acid sequences are shown in Table 1.

[0106] Table 1 Amino acid sequence

[0107]

[0108]

[0109] Among them, NC1, NC3, and NC5 introduce 3 to 5 additional Cs on the basis of NC.

[0110] 2. Construction of recombinant strains

[0111] The pColdM plasmid was constructed by using pColdM-S: CTCGAGGGATCCGAATTCA (shown in SEQ ID NO. 14) and pColdM-A: GAGCTCCATGGGCACTTTG (shown in SEQ ID NO. 15) as primers to mutate the pColdIII plasmid and introduce an NcoI site.

[0112] The gene encoding the amino acid sequence in Table 1 was synthesized, and the nucleotide sequence contained a 5' Nco I restriction site, a 5' flanking sequence GC, and a 3' Bam HI restriction site.

[0113] The synthesized genes were inserted between NcoI and BamHI of the pColdM plasmid to obtain the corresponding recombinant collagen plasmids. The recombinant plasmids were then transformed into E. coli BL21 (DE3) competent cells by the CaCl2 method. LB plates containing antibiotics were coated, cultured, and screened to obtain recombinant strains for producing hybrid collagen. According to the names of the collagens, the recombinant strains were named E. coli-mC, E. coli-CC, E. coli-NC, E. coli-NC1, E. coli-NC3, and E. coli-NC5, respectively.

[0114] 3. Collagen sample preparation

[0115] After the recombinant strain was induced to ferment, the fermentation broth was centrifuged at 8000 rpm for 5 minutes and the cells were collected separately. The cells were resuspended in 10mM phosphate buffer, the cells were broken using a high-pressure homogenizer, and then centrifuged at 4°C and 10000 rpm for 20 minutes to remove cell debris. The impurities were then filtered through a 0.45μm microporous filter membrane. The sample was injected into a 5mL His-Trap HP affinity chromatography column installed on a protein purifier, rinsed with washing solution for 8 column volumes, and then eluted with an elution buffer containing an imidazole gradient (140mM, 400mM). The peak protein was collected for SDS-PAGE electrophoresis analysis. The globular guide folding domain was then excised using trypsin at a final concentration of 0.05 mg / mL at 25°C for 8 hours. The protein was then dialyzed for desalination and freeze-dried to obtain lyophilized collagen powders. The collagens were named mC, CC, NC, NC1, NC3, and NC5 according to their sequence (the amino acid sequences of the cleaved and folded collagens are shown in SEQ ID NOs. 17 to 21). A small amount of each lyophilized powder was dissolved in water and analyzed by SDS-PAGE and MALDI-TOF.

[0116] Figure 2 The purified protein showed a single band on SDS-PAGE. Since collagen is a rod-shaped protein, the SDS-PAGE molecular weight of the globular protein marker used was larger than expected.

[0117] Figure 3 The molecular weight obtained by mass spectrometry was consistent with the theoretical molecular weight, proving that the obtained collagen molecular weight was correct.

[0118] Example 2: Determination of collagen secondary structure

[0119] The collagen proteins (mC, CC, NC, NC1, NC3, and NC5) designed and prepared in Example 1 were prepared at a concentration of 1 mg / mL and allowed to stand at 4°C for at least 24 hours. Circular dichroism spectroscopy was performed at 4°C using a 1 mm cuvette over the entire wavelength range from 190 nm to 260 nm, with a 1 nm interval and a dwell time of 5 seconds at each wavelength. Thermal shift experiments were performed at 220 nm, with the temperature equilibrated from 4°C to 80°C for 8 seconds at each temperature, at a rate of 1°C / 6 minutes. The CD spectrum of the typical collagen triple helix structure shows a positive absorption peak at 225 nm.

[0120] The results are as follows Figure 4 As shown, under full wavelength scanning, the collagen designed and prepared in Example 1 has a characteristic absorption peak near 225nm; the results of the thermal change experiment show that as the temperature increases, the characteristic absorption value at 225nm changes sharply between 37 and 55°C, which is manifested as the destruction of the secondary structure of the collagen, that is, the unwinding of the triple helix.

[0121] The results of circular dichroism and thermal change tests showed that the collagens designed in Example 1 (mC, CC, NC, NC1, NC3, NC5) can all be correctly folded to form a collagen triple helical structure and have high thermal stability.

[0122] Example 3: (PPG) 10 Effect of Cys replacement in different regions on fiber morphology

[0123] The freeze-dried collagen mC, CC, and NC prepared in Example 1 were prepared with 10 mM PB to a final concentration of 0.5 mM, or 10 mM PB containing 0.1% H2O2 and 100 mM DTT was added. After incubation at 4°C for 3.5 days, a small amount was dropped onto a copper grid, allowed to adsorb for 45 seconds, and then blotted dry with filter paper. The grid 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.

[0124] Transmission electron microscopy results are as follows Figure 5 As shown in (PPG) 10 The introduction of Cys into the motif will affect the fiber morphology after self-assembly. Under natural conditions, the light and dark striped fibers formed by CC and NC are relatively good; under reducing conditions, mC, CC and NC form obvious light and dark striped fibers because they are not affected by Cys cross-linking; under oxidizing conditions, only NC can form better light and dark striped fibers.

[0125] Example 4: (PPG) 10 Effects of Cys replacement in different regions on assembly, hydrogel mechanical properties and internal structure

[0126] The collagen mC, CC and NC prepared in Example 1 were tested for their properties, as follows:

[0127] 1. Collagen particle size detection

[0128] 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 in a buffer solution of 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 measured by dynamic light scattering. Figure 6 As shown in (a), mC, CC, and NC all aggregated into large particles with a hydration radius (Rh) of approximately 1000 nm. The particle size under oxidizing conditions was larger than that under reducing conditions, with NC forming the largest aggregate, at 1828 nm.

[0129] 2. Collagen hydrogel performance testing

[0130] (1) Hydrogel appearance and modulus

[0131] The freeze-dried collagen mC, CC and NC prepared in Example 1 were prepared into a solution with a final concentration of 100 g / L. After being placed at 4° C. for 3.5 days, 0.1% H 2 O 2 was added and cross-linked at room temperature for 2 hours.

[0132] The rheological properties of the hydrogels are shown in Figure 2. Figure 6 As shown in b, at a concentration of 10% w / v, mC, CC, and NC all behave as hydrogels, with storage modulus G' > loss modulus G", and the modulus of NC is larger than that of mC and CC. The appearance of the hydrogel is shown in Figure 7 As shown in a, b, and c, mC, CC, and NC formed a translucent hydrogel. The sample did not flow in the inverted transparent centrifuge tube, forming a collagen hydrogel.

[0133] (2) Hydrogel scanning electron microscopy

[0134] A small amount of the hydrogel prepared in step (1) was frozen with liquid nitrogen and placed in a freeze dryer to freeze-dry the sample. After spraying gold on the cross section, the internal structure of the collagen hydrogel was observed using a scanning electron microscope. The results are as follows: Figure 7 As shown in Figures d, e, f, g, h, and i, the hydrogel samples of mC, CC, and NC all appear as loose and porous collagen sponges; however, the internal structure of mC is an isotropic network-like microporous structure, while the internal structures of CC and NC are anisotropic lamellar stacking structures.

[0135] Example 5: Effect of replacing different amounts of Cys in the CL-domain on fiber morphology

[0136] 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 dried with filter paper. The grid was then negatively stained with 0.75% phosphotungstic acid for 20 seconds, dried with filter paper, and observed using a Hitachi H-7650 transmission electron microscope.

[0137] Transmission electron microscopy results are as follows Figure 8 As shown, replacing different numbers of Cys in the CL-domain affects the morphology of the self-assembled fibers. A larger number of Cys residues results in more severe fiber damage. Under natural conditions, NC1 and NC3 can form fibers with distinct light and dark stripes. Under a reducing environment, NC1 and NC3 form distinct light and dark stripes, while NC5 still cannot form light and dark stripes. Under an oxidizing environment, only NC1 can form well-proportioned light and dark stripes.

[0138] Example 6: Effects of different amounts of CL-domain replacement on mechanical properties and internal structure of adhesives

[0139] The collagen NC1, NC3 and NC5 prepared in Example 1 were tested for their properties, as follows:

[0140] 1. Collagen particle size detection

[0141] The freeze-dried collagen NC1, NC3 and NC5 prepared in Example 1 were prepared into a solution with a final concentration of 0.5 mg / mL in a buffer solution of 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 measured by dynamic light scattering.

[0142] The results are as follows Figure 9 As shown in a, NC1, NC3 and NC5 all aggregated into large particles, with NC1 having the largest hydrated particle size (Rh) of 1427 nm; NC5 having 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.

[0143] 2. Collagen hydrogel performance testing

[0144] (1) Hydrogel appearance and modulus

[0145] 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. After being placed at 4° C. for 3.5 days, 0.1% H 2 O 2 was added and cross-linked at room temperature for 2 hours.

[0146] The rheological properties of the hydrogels are shown in Figure 2. Figure 9 As shown in b, at a concentration of 10%, NC1, NC3, and NC5 all behave as hydrogels, with storage modulus G' > loss modulus G", and the more Cys is replaced in the CL-domain, 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 becomes smaller. The appearance of the hydrogel is shown in Figure 10 As shown in a, b, and c, NC1, NC3, and NC5 formed translucent hydrogels. The samples did not flow in the inverted transparent centrifuge tubes, forming collagen hydrogels. After gelation at 4°C for more than 2 weeks, NC1 could be picked up with pointed tweezers, showing strong mechanical properties, while NC3 and NC5 could not be picked up with pointed tweezers.

[0147] (2) Hydrogel scanning electron microscopy

[0148] A small amount of hydrogel was frozen with liquid nitrogen and placed in a freeze dryer to freeze-dry the sample. After spraying gold on the cross section, the internal structure of the collagen hydrogel was observed using a scanning electron microscope. Figure 10 As shown in Figures d, e, and f, the hydrogel samples of NC1, NC3, and NC5 all appear as loose and porous collagen sponges. The internal structure of NC5 is an isotropic network-like microporous structure, while the internal structures of NC1 and NC3 are anisotropic lamellar stacking structures.

[0149] Example 7: Verification of the biocompatibility and bioactivity of the designed type I collagen

[0150] 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, and bovine serum albumin (BSA) and rat tail type I collagen at the same concentration were used as negative and positive controls.

[0151] 100 μL of collagen solution was added to three replicates of each 96-well plate. After adsorption at 4°C for 24 hours, the solution was aspirated and allowed to dry overnight before sterilization under UV irradiation for 30 minutes. Finally, the plates were blocked with 5% BSA for 2 hours. 2000 MC-3T3-E1 cells were resuspended in DMEM supplemented with 10% FBS per well and 100 μL was seeded onto the plates. Cell viability was measured using a CCK8 assay kit at 0, 1, 3, and 5 days.

[0152] The results are as follows Figure 11 As shown in a, the designed collagen had a significant effect on cell proliferation and no cytotoxicity. In the same way, collagen was adsorbed onto a 96-well plate and 10,000 cells were seeded. After culturing for 6 hours, the cells were washed three times with 10mM PBS and then the cell viability was determined using a CCK8 kit. Figure 11 b. Figure 11 As shown in c, compared with samples with poor fiber morphology, NC1 showed a significant promoting effect on cell adhesion, and was better than natural Type I collagen. The expression level of VCL mRNA related to the cytoskeletal protein Vinculin was also significantly increased. Figure 11 As shown in Figure 5, when the same method was used to evaluate the cell spreading area and phalloidin was used for cytoskeleton staining, cells adhered to the NC1 matrix showed a larger spreading area and more obvious cytoskeleton extension, which was comparable to Type I. The results showed that the synthetic collagen NC1 has excellent biocompatibility.

[0153] Furthermore, we verified the osteogenic differentiation ability of MC3T3-E1 osteoblast precursor cells on the designed collagen fiber surface. After one week of induction of MC3T3-E1 osteogenic differentiation, the osteogenic differentiation ability was detected by alkaline phosphatase (ALP) staining. Figure 11 As shown in (fi), the ALP staining area and ALP activity of NC and NC1 were comparable to or even greater than those of type I collagen, and significantly exceeded those of mC, CC, NC3, NC5, or BSA sample groups.

[0154] At the same time, type I collagen fibers NC and NC1 induced the expression of osteoblast-specific transcription factor RUNX2 and bone matrix protein gene type I collagen α1 chain Col1a1, and their levels exceeded those of other synthetic collagens and even higher than those of natural type I collagen. As described in the review, it was demonstrated that NC and NC1 collagens can promote the osteogenic differentiation of MC3T3-E1 osteoblast precursor cells.

[0155] Example 8: Verification of fracture repair function of NC1 collagen hydrogel

[0156] The freeze-dried 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 an NC1 collagen hydrogel, and its fracture repair function was verified.

[0157] Six-week-old male Sprague-Dawley rats were selected for this study. A 1.5-2 cm incision was made on the lateral side of the femur. Blunt dissection of the mucosa and muscle was performed to expose the mid-femoral segment. Intramedullary fixation was performed using a 1.2 mm Kirschner wire, and the fracture ends were secured with 3-0 absorbable sutures. Postoperatively, the wound was irrigated with 0.9% sodium chloride solution, and the muscle and skin were sutured. Penicillin (100,000 units / rat) was administered intramuscularly. This injection was continued for three consecutive days postoperatively to prevent infection.

[0158] A blank control group (normal saline), an NC1 collagen hydrogel, and a positive control group (rat tail type I collagen hydrogel) were injected 200 μL into the fracture area every other day for two weeks. Four weeks later, the rats were sacrificed and fracture repair was observed using micro-CT. The remaining samples were decalcified, sectioned, and stained with H&E and immunohistochemically (VEGF, RUNX2, and ALP) to assess angiogenesis and the expression of proteins involved in bone synthesis.

[0159] The results are as follows Figure 12 As shown in the figure, micro-CT results showed that compared with the untreated model, the NC1 collagen hydrogel exhibited a significant fracture repair effect, which was very close to that of the rat tail native type I hydrogel. Analysis of bone-related parameters also showed that bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number, and connective tissue density were significantly increased in the fracture area treated with the NC1 collagen hydrogel.

[0160] H&E staining results showed no obvious inflammatory cell infiltration, and no significant difference was found among the groups. Figure 12 c, The vascular endothelial growth factor (VEGF) in the fracture area of SD rats in the NC1 collagen hydrogel-treated group was significantly increased, indicating that angiogenesis in the local microenvironment of the fracture was active and that NC1 collagen hydrogel had the ability to promote angiogenesis.

[0161] Compared with the blank group, the protein expression level of Runx2, a core transcription factor for osteogenic differentiation, and the alkaline phosphatase (ALP) activity in the NC1 collagen hydrogel group were significantly increased (p<0.0005), and were higher than those in rat tail collagen type I hydrogel.

[0162] The above 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.

[0163] The amino acid sequence used in the present invention is:

[0164] Amino acid sequence of V-domain (SEQ ID NO.1):

[0165] ADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD

[0166] Amino acid sequence of CL-domain (SEQ ID NO. 3):

[0167] PRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQG

[0168] Nucleotide sequence of the CL-domain (SEQ ID NO.13)

[0169] CCTCGTGGTGAACAGGGTCCGCAGGGCCTGCCGGGTAAAGATGGCGAAGCCGGTGCCCAGGGCCCGGCAGGTCCTCGTGGTCCTCAGGGTCCGCAAGGTCTGCCGGGTCCGCAGGGTCCTGCCGGTCCTATGGGTCCGGCCGGTTTTCCGGGTGAACGTGGTGAAAAAGGTGAACCGGGTACCCAGGGCGCCAAAGGTGACCGTGGTGAAACCGGTCCGGTTGGCCCGCGTGGTGAACGCGGTGAAGCAGGCCCGGCCGGTAAAGATGGTGAACGTGGCCCGGTTGGTCCGGCAGGTCCGAGAGGTCCGCAGGGACCTCAGGGTCTGCCGGGACCTCAGGGCCCTGCAGGTGCTCAGGGT

[0170] Nucleotide sequence of the V-domain (SEQ ID NO.16)

[0171] GCCGATGAACAGGAAGAAAAAGCCAAAGTGCGTACCGAACTGATTCAGGAACTGGCACAGGGCCTGGGCGGTATTGAAAAGAAAAATTTTCCGACCCTGGGTGACGAAGATCTGGATCATACCTATATGACCAAACTGCTGACCTATCTGCAGGAACGTGAACAGGCCGAAAATAGTTGGCGTAAACGTCTGCTGAAAGGCATTCAGGATCATGCACTGGAT

[0172] mC after folding and digestion (SEQ ID NO.17)

[0173] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQGPPGPPGPPGPPGPPGCPGPPGPPGPPGPPG Folded and digested CC (SEQ ID NO.18)

[0174] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGPRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGACGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPC

[0175] Folded and digested NC (SEQ ID NO.19)

[0176] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGCRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPC

[0177] Folded and digested NC1 (SEQ ID NO.20)

[0178] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGCRGEQGPQGLPGKDGEAGAQGPAGPRGPQGPQGLPGPQGPAGPMGPAGFPGERGEKGECGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPC

[0179] Folded and digested NC3 (SEQ ID NO.21)

[0180] PPGPPGPPGPPGPPGPPGPPGPPGPPGPPGCRGEQGPQGLPGKDGEAGAQGPAGPRGPCGPQGLPGPQGPAGPMGPAGFPGERGEKGECGTQGAKGDRGETGPVGPRGERGEAGPCGKDGERGPVGPAGPRGPQGPQGLPGPQGPAGAQGPPGPPGPPGPPGPPGPPGPPGPPGPPGPPC

[0181] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

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

2. The collagen single chain according to claim 1, characterized in that One cysteine was introduced at the front and end of the second repeat sequence; Alternatively, one cysteine is introduced at the end of each of the first and second repeat sequences.

3. The collagen single chain according to any one of claims 1 to 2, characterized in that Introducing 1 to 3 cysteines into the collagen domain; Optionally, a cysteine is introduced into the collagen domain; Optionally, two cysteines are introduced into the collagen domain, with the two cysteines separated by 40 to 80 amino acids; Optionally, three cysteines are introduced into the collagen domain, and the three cysteines are located in amino acids 1 to 40, 41 to 80, and 81 to 120 of the collagen domain, respectively.

4. The collagen single chain according to any one of claims 1 to 3, characterized in that A folding domain is connected 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; Optionally, the amino acid sequence of the first and second repeat sequences is (PPG) n , n is an integer from 4 to 30; Optionally, the collagen domain includes human collagen, bacterial collagen, recombinant human collagen, or recombinant bacterial collagen; Optionally, the amino acid sequence of the collagen domain is shown in SEQ ID NO. 3; Optionally, there is a connecting peptide between the folding domain and the repeat sequence, and the amino acid sequence is shown in SEQ ID NO.

4.

5. The collagen single chain according to any one of claim 4, 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; Optionally, the amino acid sequence of the collagen single chain after enzymatic cleavage and folding is as shown in any one of SEQ ID NO.18 to SEQ ID NO.

21.

6. A gene encoding the single-chain collagen protein according to any one of claims 1 to 5, or a plasmid or cell carrying the gene encoding the single-chain collagen protein according to any one of claims 1 to 5; Optionally, the plasmid includes the pColdIII series or the pET series.

7. The collagen product prepared from the collagen single chain according to any one of claims 1 to 5; Optionally, the collagen product includes type I collagen, collagen fibers and collagen hydrogel.

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

9. The method according to claim 8, characterized in that One cysteine was introduced at the front and end of the second repeat sequence; Alternatively, one cysteine is introduced at each end of the first repeat sequence and the second repeat sequence; Optionally, 1 to 3 cysteines are introduced into the collagen domain; Optionally, a cysteine is introduced into the collagen domain; Optionally, two cysteines are introduced into the collagen domain, with the two cysteines separated by 40 to 80 amino acids; Optionally, three cysteines are introduced into the collagen domain, and the three cysteines are located in amino acids 1 to 40, 41 to 80, and 81 to 12 of the collagen domain, respectively.

10. Use of the collagen single chain according to any one of claims 1 to 5, or the gene, plasmid, cell according to claim 6, or the collagen product according to claim 7, or the method according to any one of claims 8 to 9 in the fields of biology, food, chemical engineering, medicine, biomaterials, tissue engineering, or cosmetics.

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