Anti-degradation recombinant collagen as well as preparation method and application thereof

Abstract

The invention discloses an anti-degradation recombinant collagen as well as a preparation method and application thereof, and belongs to the technical field of collagen and bioengineering. According to the invention, full-length or partial genes of natural collagen in a human body are selected for certain optimization combination, a collagen amino acid sequence with strong stability and high biological activity is designed, and the amino acid sequence is completely consistent with a sequence of a corresponding region of the natural collagen in theory, and has relatively low immunogenicity; and the collagen has typical biological functions of human body collagen. The recombinant collagen prepared by the invention has excellent stability, biological activity and safety.

Description

Technical Field

[0001] The invention relates to degradation-resistant recombinant collagen and a preparation method and application thereof, belonging to the technical field of collagen and bioengineering. Background Art

[0002] As a medical biomaterial, collagen has shown its unique advantages in the medical field, among which the most prominent feature is its low immunogenicity. This feature enables collagen to significantly reduce the risk of immune rejection when applied to the human body, thereby ensuring the safety and effectiveness of the treatment process. It also has broad application prospects as an implant material, tissue repair material, and drug carrier.

[0003] Collagen also has good biocompatibility. In the interaction with host cells and tissues, collagen can quickly integrate into the matrix around cells, providing strong support for cell growth and differentiation. Collagen can also promote information transmission and material exchange between cells, thus helping to maintain the normal physiological functions of cells. This biocompatibility enables collagen to play a more stable and lasting role in medical applications.

[0004] In addition to good biocompatibility and low immunogenicity, collagen has excellent mechanical properties and structural stability, and has a wide range of application value in the field of biomechanics. Its high strength allows it to withstand various forms of external forces, including pressure, tensile force, etc., without obvious deformation or structural damage. This property makes collagen one of the key factors in maintaining the structural integrity and functional stability of organisms. Whether in skin, bones or muscles and other tissues, collagen can effectively resist the influence of the external environment and maintain the stability of the morphology and function of the tissue.

[0005] Existing patents for expressing recombinant collagen in yeast are mostly concentrated on expressing humanized collagen sequences, such as "Genetically Recombinant Human Collagen Fusion Peptide Segment and Preparation Method and Application thereof" (Application No. CN201010527766.7), "A Recombinant Human Type I Collagen and Its Immobilized Fermentation Production Method" (Application No. CN201510535383.7), and "Recombinant Human Type III Collagen α1 Chain and Its Application" (Application No. CN201911093124.8), most of which are expressions of collagen fragments or fusion expressions of different fragments. In addition, some involve the expression of artificially designed collagen-like sequences, such as "Human-like collagen gene, tandem gene with different repeat numbers, recombinant plasmid containing tandem gene and preparation method" (application number CN200610098297.5), the proteins expressed are all linear single chains, and inevitably there are some sites that are easy to deamidate, hydrolyze or oxidize; at the same time, the human-like collagen sequence involved in patent CN200610098297.5 is artificially designed, but the design ideas are not explained, and the contribution of artificially designed sequences to product stability is not clear. "High-stability recombinant collagen, construction method and application thereof" (application number: CN202210954027.9) obtains a recombinant collagen sequence with improved stability by mutating the amino acid sequence, thereby improving the protein yield and storage stability. Although the high stability of the sample is guaranteed, it involves mutations in the amino acid sequence, which poses a safety risk.

[0006] The above-mentioned technologies involve recombinant collagen, which may increase unstable factors in the solution or during use, and may lead to the production of process-related impurities and uncontrollable degradation products, increase the toxicity of unknown substances, and increase immunogenicity. In addition, although some patents have improved stability by mutating collagen amino acids, the mutated collagen sequence is inconsistent with the human collagen sequence, and there is a possibility that it will be identified as a foreign body during use, which will increase the risk of immunogenicity and have certain safety risks. Other uncontrollable risks also increase, leading to an increase in the risk of clinical application.

[0007] Therefore, there is an urgent need to develop a collagen based on natural human collagen, without amino acid mutation, which is safe, has strong anti-degradation properties and biological activity and has extremely high practical and economic value. Summary of the invention

[0008] In order to solve the problems that collagen is easily degraded in vitro, has increased immunogenicity, low biological activity, increased difficulty in purification and storage, and the degradation that occurs during the application process will lead to increased difficulty in product quality control; the present invention selects the full-length or partial gene of natural collagen in the human body, performs certain optimization combinations, and designs a collagen amino acid sequence with strong stability and high biological activity. The amino acid sequence is theoretically completely consistent with the corresponding regional sequence of natural human collagen, has low immunogenicity, and maintains the typical biological functions of human collagen.

[0009] The first object of the present invention is to provide a recombinant collagen single chain, wherein the collagen single chain contains repeated collagen monomer segments;

[0010] The amino acid sequence of the collagen monomer fragment is shown in any one of SEQ ID NOs. 2 to 7.

[0011] In one embodiment, the collagen single chain contains 2 to 10 collagen monomer fragments;

[0012] Optionally, the collagen single chain contains 3 to 7 collagen monomer fragments;

[0013] Optionally, the collagen single chain contains a collagen monomer fragment with 3 to 7 amino acid sequences as shown in SEQ ID NO.7.

[0014] In one embodiment, the recombinant collagen single chain is obtained by cutting and splicing human type III collagen;

[0015] Optionally, the amino acid sequence of the human type III collagen is as shown in SEQ ID NO.1.

[0016] In one embodiment, the recombinant collagen single chain is composed of a collagen monomer fragment as shown in SEQ ID NO.2 repeated 2 to 10 times; wherein the collagen monomer fragment as shown in SEQ ID NO.2 is obtained by sequentially combining the amino acids at positions 274 to 302, 432 to 464 and 1083 to 1095 of human type III collagen (amino acid sequence as shown in SEQ ID NO.1);

[0017] Preferably, it is repeated 3 to 7 times; more preferably, it is repeated 4 to 5 times.

[0018] In one embodiment, the recombinant collagen single chain is composed of a collagen monomer fragment as shown in SEQ ID NO.3 repeated 2 to 10 times; wherein the collagen monomer fragment as shown in SEQ ID NO.3 is obtained by sequentially combining the amino acids 274 to 302, 432 to 464 and 1083 to 1095 of human type III collagen (amino acid sequence as shown in SEQ ID NO.1), and replacing the amino acid RGE therein with KGD;

[0019] Preferably, it is repeated 3 to 7 times; more preferably, it is repeated 5 to 7 times.

[0020] In one embodiment, the recombinant collagen single chain is composed of a collagen monomer fragment as shown in SEQ ID NO.4 repeated 2 to 10 times; wherein the collagen monomer fragment as shown in SEQ ID NO.4 is obtained by sequentially combining the amino acids 648 to 686 and 1093 to 1094 of human type III collagen (amino acid sequence as shown in SEQ ID NO.1);

[0021] Preferably, the step is repeated 3 to 7 times; more preferably, the step is repeated 6 to 8 times;

[0022] More preferably, on the basis of being repeated 6 to 8 times, the amino acid sequence at positions 648 to 686 is additionally connected to the C-terminus of the sequence.

[0023] In one embodiment, the recombinant collagen single chain is composed of a collagen monomer fragment as shown in SEQ ID NO.5 repeated 2 to 10 times; wherein the collagen monomer fragment as shown in SEQ ID NO.5 is obtained by sequentially combining the amino acids at positions 273 to 296, positions 432 to 464, positions 909 to 944, positions 1038 to 1061 and positions 1083 to 1106 of human type III collagen (amino acid sequence as shown in SEQ ID NO.1);

[0024] Preferably, it is repeated 3 to 7 times; more preferably, it is repeated 2 to 4 times.

[0025] In one embodiment, the recombinant collagen single chain is composed of a collagen monomer fragment as shown in SEQ ID NO.6 repeated 2 to 10 times; wherein the collagen monomer fragment as shown in SEQ ID NO.6 is obtained by sequentially combining the 648th to 686th, 909th to 944th, 1038th to 1061st, and 648th to 686th amino acids of human type III collagen (amino acid sequence as shown in SEQ ID NO.1);

[0026] Preferably, repeat 3 to 7 times; more preferably, repeat 3 to 5 times;

[0027] More preferably, on the basis of repeating 3 to 5 times, the amino acid sequence at positions 648 to 686 is additionally connected to the C-terminus of the sequence.

[0028] In one embodiment, the recombinant collagen single chain is composed of a collagen monomer fragment as shown in SEQ ID NO.7 repeated 3 to 7 times; wherein the collagen monomer fragment as shown in SEQ ID NO.7 is obtained by sequentially combining the 618th to 686th, 1170th to 1190th, and 1083th to 1095th amino acids of human type III collagen (amino acid sequence as shown in SEQ ID NO.1);

[0029] Preferably, the process is repeated 3 to 7 times; more preferably, the process is repeated 3 to 5 times.

[0030] In one embodiment, a 6His tag may be connected to the C-terminus of the recombinant collagen single chain for protein purification and separation.

[0031] The second object of the present invention is to provide a gene encoding any of the above-mentioned collagen single chains.

[0032] The present invention also provides a plasmid or a cell carrying a gene encoding any of the above-mentioned single-chain collagens.

[0033] In one embodiment, the plasmid is a plasmid for prokaryotic expression, including but not limited to pET series vectors, pGEX series vectors, pBAD series vectors, and pUC series vectors;

[0034] Optionally, the plasmid includes pET-28a, pET-32a, pGEX-4T-1, pBAD / Myc-His, pUC19.

[0035] In one embodiment, the plasmid is a eukaryotic expression plasmid, including but not limited to pcDNA series vectors, pCMV series vectors, pIRES series vectors, pEGFP series vectors, pYES series vectors, and pPIC series vectors;

[0036] Optionally, the plasmid includes pcDNA3.1, pCMV-Tag, pCMV-Sport, pIRES2-EGFP, pEGFP-C1, pYES2, pPICZ, pPIC3.5.

[0037] In one embodiment, the cell is a host cell, including but not limited to prokaryotic cells, such as Escherichia coli and Bacillus subtilis; yeast cells, such as Pichia pastoris and Saccharomyces cerevisiae; mammalian cells, such as HEK293 cells and CHO cells.

[0038] The third object of the present invention is to provide the use of any of the above-mentioned recombinant collagen single chains, the above-mentioned genes, the above-mentioned plasmids or cells in the preparation of collagen products.

[0039] In one embodiment, the collagen product includes food, health care products, medicines, medical devices or cosmetics;

[0040] Optionally, the food includes functional food and food additives;

[0041] Optionally, the medicine includes a wound repair material, a cartilage repair material, and a drug delivery carrier;

[0042] Optionally, the medical device includes tissue engineering materials, surgical auxiliary equipment, and oral repair materials;

[0043] Optionally, the cosmetics include skin care products, hair care products, and anti-aging products.

[0044] In one embodiment, the functional food includes but is not limited to collagen peptide drinks, such as juice collagen drinks, collagen-containing coffee; collagen candies, such as collagen tablet candies, collagen soft candies;

[0045] Food additives include, but are not limited to, collagen peptides, which are used to improve food texture and flavor; thickeners, which are used for thickening and gelling; and collagen casings, which are used to prepare processed products such as sausages.

[0046] In one embodiment, the health care products include but are not limited to collagen oral liquid, collagen tablets, collagen powder, collagen joint health soft capsules, and bone health drinks.

[0047] In one embodiment, the medicine includes, but is not limited to, wound repair materials, such as collagen sponge (to promote wound healing), collagen hemostatic dressing (to stop bleeding quickly), and collagen membrane (for postoperative repair);

[0048] Cartilage repair materials, such as collagen injections (repair of articular cartilage defects), collagen-based cartilage transplant materials;

[0049] Drug delivery vehicles, such as collagen microspheres (encapsulating active ingredients for sustained release of drugs), collagen gel injections (local anti-inflammatory or anti-tumor drug delivery).

[0050] In one embodiment, the medical device includes, but is not limited to, tissue engineering materials, such as collagen scaffolds (for cell culture and tissue regeneration), collagen-based composite materials (for bone or soft tissue repair), and collagen filling materials (for filling tissues or filling cavities);

[0051] Surgical auxiliary equipment includes but is not limited to collagen sutures (degradable sutures) and collagen hemostatic sponges (intraoperative hemostasis);

[0052] Oral restoration materials include but are not limited to collagen membranes (alveolar bone filling) and collagen periodontal regeneration materials.

[0053] In one embodiment, the cosmetics include skin care products, such as collagen masks (moisturizing, repairing, anti-aging), collagen essences (enhancing skin elasticity), and collagen lotions (hydrating and locking in moisture);

[0054] Hair products, such as collagen shampoo (repairs damaged hair) and collagen conditioner (replenishes nutrients and softens hair);

[0055] Anti-aging products, such as collagen anti-wrinkle cream (to improve skin firmness) and collagen freeze-dried powder (to promote skin regeneration and repair fine lines).

[0056] The fourth object of the present invention is to provide a collagen product, said product containing any of the above-mentioned recombinant collagen single chains;

[0057] Optionally, the collagen product includes food, health care product, medicine, medical device or cosmetic.

[0058] Optionally, the food includes functional food and food additives;

[0059] Optionally, the medicine includes a wound repair material, a cartilage repair material, and a drug delivery carrier;

[0060] Optionally, the medical device includes tissue engineering materials, surgical auxiliary equipment, and oral repair materials;

[0061] Optionally, the cosmetics include skin care products, hair care products, and anti-aging products.

[0062] In one embodiment, the functional food includes but is not limited to collagen peptide drinks, such as juice collagen drinks, collagen-containing coffee; collagen candies, such as collagen tablet candies, collagen soft candies;

[0063] Food additives include, but are not limited to, collagen peptides, which are used to improve food texture and flavor; thickeners, which are used for thickening and gelling; and collagen casings, which are used to prepare processed products such as sausages.

[0064] In one embodiment, the health care products include but are not limited to collagen oral liquid, collagen tablets, collagen powder, collagen joint health soft capsules, and bone health drinks.

[0065] In one embodiment, the medicine includes, but is not limited to, wound repair materials, such as collagen sponge (to promote wound healing), collagen hemostatic dressing (to stop bleeding quickly), and collagen membrane (for postoperative repair);

[0066] Cartilage repair materials, such as collagen injections (to repair articular cartilage defects) and collagen-based cartilage transplant materials;

[0067] Drug delivery vehicles, such as collagen microspheres (encapsulating active ingredients for sustained release of drugs), collagen gel injections (local anti-inflammatory or anti-tumor drug delivery).

[0068] In one embodiment, the medical device includes, but is not limited to, tissue engineering materials, such as collagen scaffolds (for cell culture and tissue regeneration), collagen-based composite materials (for bone or soft tissue repair), and collagen filling materials (for filling tissues or filling cavities);

[0069] Surgical auxiliary equipment includes but is not limited to collagen sutures (degradable sutures) and collagen hemostatic sponges (intraoperative hemostasis);

[0070] Oral restoration materials include but are not limited to collagen membranes (alveolar bone filling) and collagen periodontal regeneration materials.

[0071] In one embodiment, the cosmetics include skin care products, such as collagen masks (moisturizing, repairing, anti-aging), collagen essences (enhancing skin elasticity), and collagen lotions (hydrating and locking in moisture);

[0072] Hair products, such as collagen shampoo (repairs damaged hair) and collagen conditioner (replenishes nutrients and softens hair);

[0073] Anti-aging products, such as collagen anti-wrinkle cream (to improve skin firmness) and collagen freeze-dried powder (to promote skin regeneration and repair fine lines).

[0074] The present invention also provides a collagen hydrogel, characterized in that it is prepared using any of the above-mentioned recombinant collagen single chains, and the preparation method comprises:

[0075] The recombinant collagen single chain and the cross-linking agent are mixed, and the collagen hydrogel is obtained by cross-linking reaction;

[0076] Optionally, the cross-linking agent includes PEG-NHS activated lipid, EDC-NHS, glutaraldehyde;

[0077] Optionally, the cross-linking reaction is carried out at 35-40° C. for 20-60 min;

[0078] Optionally, the concentration ratio of the cross-linking agent to the recombinant collagen single chain is 1:20 to 1:100.

[0079] The fifth object of the present invention is to provide a method for improving the performance of recombinant collagen single chain, wherein the performance includes one or more of stability, biological activity, and safety;

[0080] The method comprises:

[0081] Using repeated collagen monomer segments to form a single chain of recombinant collagen;

[0082] Wherein, the amino acid sequence of the collagen monomer fragment is shown in any one of SEQ ID NOs. 2 to 7;

[0083] Optionally, the collagen single chain contains 2 to 10 collagen monomer fragments;

[0084] Optionally, the collagen single chain contains a collagen monomer fragment with 3 to 7 amino acid sequences as shown in SEQ ID NO.7.

[0085] Beneficial Effects of the Invention

[0086] The present invention selects the full-length or partial gene of natural collagen in the human body, performs certain optimization combinations, and designs a collagen amino acid sequence with strong stability and high biological activity. The amino acid sequence is theoretically completely consistent with the corresponding region sequence of natural human collagen, has low immunogenicity, and has the typical biological functions of human collagen.

[0087] Specifically,

[0088] (1) The recombinant collagen prepared by the present invention has excellent stability. After being heated at 60°C for 10 days and 121°C for 20 minutes, it still maintains structural and purity stability, and has good acid-base (pH 6-8) tolerance and freeze-thaw (-20°C, -80°C, three cycles) stability;

[0089] (2) The adhesion activity and proliferation activity of the recombinant collagen prepared by the present invention are high, which are close to the adhesion activity of bovine collagen, with no significant difference;

[0090] (3) After incubating cells for 24 hours, the recombinant collagen prepared by the present invention has a substantially intact cell morphology, a cell viability value of 97.6%, no cytotoxicity, and high biocompatibility;

[0091] (4) The hydrogel prepared based on the recombinant collagen of the present invention does not change in appearance before and after heat treatment (121°C, 20 min) and has little effect on dynamic viscosity. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Schematic diagram of the recombinant plasmid;

[0093] Figure 2 This is the SDS-PAGE test result of the supernatant of recombinant collagen expressed by genetically engineered Pichia pastoris;

[0094] Figure 3 The results of SDS-PAGE test of the supernatant of large-scale fermentation are shown;

[0095] Figure 4 This is the SDS-PAGE test result of recombinant collagen after purification and lyophilization;

[0096] Figure 5 This is the HPLC test result of recombinant collagen after purification and lyophilization;

[0097] Figure 6 This is the appearance of the recombinant collagen purified freeze-dried sample;

[0098] Figure 7 The results of the stability test of recombinant collagen; A is the thermal stability result of the recombinant collagen single chain JU-3-3; B is the thermal stability result of the competitor; C is the thermal stability result of JU-4-3, JU-4-6, JU-4-7, and JU-4-8;

[0099] Figure 8 This is the result of the recombinant collagen cell adhesion activity test;

[0100] Fig. 9 This is the result of the recombinant collagen cell proliferation activity test;

[0101] Fig.10 This is the result of the cytotoxicity test of recombinant collagen;

[0102] Fig.11 The results of the acid-base tolerance and freeze-thaw stability tests of recombinant collagen;

[0103] Fig.12 This is the appearance structure of recombinant collagen hydrogel before and after heat treatment;

[0104] Fig.13 is the dynamic viscosity of recombinant collagen hydrogel before and after heat treatment. DETAILED DESCRIPTION

[0105] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0106] The raw materials used in the examples are:

[0107] Seed culture medium YPG (yeast powder 10 g / L, peptone 20 g / L, glycerol 10 g / L);

[0108] Fermentation medium (glycerol 40 g / L, magnesium sulfate heptahydrate 9 g / L, calcium sulfate dihydrate 0.8 g / L, potassium sulfate 10 g / L, ammonium dihydrogen phosphate 25 g / L, PTM1 trace element 4 ml / L); feed medium (50% W / V glycerol, 12 ml PTM1 trace element per liter);

[0109] Induction medium (100% methanol, 12 mL PTM1 trace elements per liter); PTM1: sterilize by filtering with a 0.22 μm filter membrane and store at 4°C. After the fermentation medium is sterilized at high temperature, add PTM1 when the temperature drops to room temperature, and adjust the pH to 4 with ammonia water;

[0110] Buffer A: 25 mM C6H8O7-Na3C6H5O7 (citric acid-sodium citrate) buffer, pH 5.0;

[0111] Buffer B: 25 mM C6H8O7-Na3C6H5O7 (citric acid-sodium citrate) + 1 M NaCl, pH 5.0.

[0112] pPICzα empty vector was purchased from Thermo Fisher Scientific;

[0113] SacⅠ was purchased from Thermo Fisher.

[0114] Example 1: Design and expression of recombinant collagen

[0115] 1. Design collagen sequence

[0116] Based on the sequence of human type III collagen (https: / / www.uniprot.org / uniprot / P02461; COL3A1, the amino acid sequence is shown in SEQ ID NO.1), recombinant collagen was designed as follows:

[0117] (1) The amino acids at positions 274 to 302, 432 to 464, and 1083 to 1095 were sequentially combined to obtain a recombinant collagen monomer fragment 1, the amino acid sequence of which is shown in SEQ ID NO. 2. SEQ ID NO. 2 was repeated 6 times and a 6His tag was connected to the C-terminus to obtain a recombinant collagen single chain, named JU-4-2, the amino acid sequence of which is shown in SEQ ID NO. 8.

[0118] (2) Based on the recombinant collagen monomer fragment 1 (the amino acid sequence is shown in SEQ ID NO.2), RGE was replaced with KGD to obtain the recombinant collagen monomer fragment 2, the amino acid sequence of which is shown in SEQ ID NO.3. SEQ ID NO.3 was repeated 6 times and a 6His tag was connected to the C-terminus to obtain a recombinant collagen single chain, named JU-4-3, the amino acid sequence of which is shown in SEQ ID NO.9.

[0119] (3) The amino acid combinations at positions 648 to 686 and 1093 to 1094 were sequentially selected to obtain recombinant collagen monomer fragment 3, the amino acid sequence of which is shown in SEQ ID NO.4. SEQ ID NO.4 was repeated 7 times and positions 648 to 686 and a 6His tag were connected to the C-terminus to obtain a recombinant collagen single chain, named JU-4-6, the amino acid sequence of which is shown in SEQ ID NO.10.

[0120] (4) The amino acid combinations at positions 273 to 296, 432 to 464, 909 to 944, 1038 to 1061 and 1083 to 1106 were sequentially selected to obtain recombinant collagen monomer fragment 4, whose amino acid sequence is shown in SEQ ID NO.5. SEQ ID NO.5 was repeated three times and a 6His tag was connected to the C-terminus to obtain a recombinant collagen single chain, named JU-4-7, whose amino acid sequence is shown in SEQ ID NO.11.

[0121] (5) The amino acid combinations at positions 648 to 686, 909 to 944, 1038 to 1061, and 648 to 686 were sequentially selected to obtain recombinant collagen monomer fragment 5, the amino acid sequence of which is shown in SEQ ID NO. 6. SEQ ID NO. 6 was repeated 4 times and the sequence at positions 648 to 686 and a 6His tag were connected to the C-terminus to obtain a recombinant collagen single chain, named JU-4-8, the amino acid sequence of which is shown in SEQ ID NO. 12.

[0122] (6) The amino acids at positions 618 to 686, 1170 to 1190, and 1083 to 1095 were sequentially combined to obtain recombinant collagen monomer fragment 6, the amino acid sequence of which is shown in SEQ ID NO.7. SEQ ID NO.7 was repeated 4 times and a 6His tag was connected to the C-terminus to obtain a recombinant collagen single chain, named JU-3-3, the amino acid sequence of which is shown in SEQ ID NO.13.

[0123] 2. Construction of recombinant plasmid and recombinant bacteria

[0124] Take the amino acid sequences SEQ ID NO.8-13 designed in step 1, entrust Nanjing GenScript Biotech Co., Ltd. to complete the corresponding nucleotide sequences (excluding 6His tags, corresponding to nucleotide sequences SEQ ID NO.14-19, respectively), and insert them into the α-factor reading frame of the pPICzα empty vector (purchased from Thermo Fisher Scientific) to obtain recombinant plasmids, respectively named pPICzα-JU-4-2 (amino acid sequence SEQ ID NO.8, nucleotide sequence SEQ ID NO.14), pPICzα-JU-4-3 (expressing amino acid sequence SEQ ID NO.9, nucleotide sequence SEQ ID NO.15), pPICzα-JU-4-6 (expressing amino acid sequence SEQ ID NO.10, nucleotide sequence SEQ ID NO.16), pPICzα-JU-4-7 (expressing amino acid sequence SEQ ID NO.11, nucleotide sequence SEQ ID NO.17), pPICzα-JU-4-8 (expressing amino acid sequence SEQ ID NO.12, nucleotide sequence SEQ ID NO.18) and pPICzα-JU-3-3 (expressing amino acid sequence SEQ ID NO.13, nucleotide sequence SEQ ID NO.19), the plasmid schematic diagram is shown in Figure 1 shown.

[0125] The above-mentioned recombinant plasmid (3 μg) was digested with SacⅠ (purchased from Thermo Fisher) at 37°C for 2 h, linearized, recovered, and electroporated into Pichia pastoris GS115 competent cells; the electroporated bacterial solution was spread on YPD / Zeocin plates and inverted cultured at 30°C for 2 to 5 days, and single colonies were selected to verify the Pichia pastoris genetic engineering, and named P. pastoris-JU-4-2, P. pastoris-JU-4-3, P. pastoris-JU-4-6, P. pastoris-JU-4-7, P. pastoris-JU-4-8, and P. pastoris-JU-3-3, respectively.

[0126] 3. Induced expression and identification of recombinant collagen

[0127] The Pichia yeast genetic engineering obtained in 2 was inoculated into 50 mL centrifuge tubes containing 5 mL BMGY medium and cultured at 28-30°C and 220 rpm until OD 600 The cells were collected by centrifugation at 3000 g for 5 min at room temperature and resuspended in BMMY medium to an OD of 10. 600The culture medium was placed on a shaker at 30°C and 220 rpm for 3 days, 100% methanol was added to the culture medium every 24 hours to a final concentration of 1.0%, and bacterial samples were collected at 48h and 72h after methanol induction. The sample volume was 1 mL and placed in a 1.5 mL EP tube. The tube was centrifuged at 12000 g for 5 min at 4°C to collect the expression supernatant, and the samples to be tested were stored at -80°C for later use.

[0128] The results of SDS-PAGE test of supernatant were Figure 2 As shown, the results showed that all six collagen sequences could be secreted into the culture supernatant.

[0129] Example 2: Preparation of recombinant collagen by fermentation

[0130] 1. Fermentation to prepare recombinant collagen

[0131] The genetically engineered Pichia pastoris P. pastoris-JU-4-2, P. pastoris-JU-4-3, P. pastoris-JU-4-6, P. pastoris-JU-4-7, P. pastoris-JU-4-8, and P. pastoris-JU-3-3 prepared in Example 1 were used for high-density fermentation to express recombinant collagen on a large scale, using a fed-batch culture method at a culture temperature of 30° C., and the steps were as follows:

[0132] (1) Pichia pastoris was inoculated into 1 L shake flasks containing 200 mL of seed medium YPG and cultured at 220 rpm and 30 °C for 22-24 h until OD 600 Reach 8-12;

[0133] (2) Use a 5L fermentation tank, fill it with 2L fermentation medium, adjust the speed to 600rpm, ventilation to 3L / min, temperature to 30°C before inoculation, adjust the pH to 4 with an alkali solution prepared with concentrated ammonia water; add 200mL of the prepared seed solution into the tank (flame circle inoculation), click the dissolved oxygen electrode to calibrate to 100%, and start fermentation after calibration;

[0134] (3) When the dissolved oxygen drops to 30% for the first time during growth, use the dissolved oxygen cascade speed function to maintain 30%; wait for the glycerol to be consumed, the dissolved oxygen rebounds, and the dissolved oxygen is greater than 60% (OD 600Value is about 50), start glycerol feeding, set the speed to 40mL / h, stop feeding after about 250mL of glycerol is added, wait for the dissolved oxygen to rise to above 60%, and perform methanol induction, set the feeding speed to 6mL / h, increase to 10mL / h after two hours, and finally increase to 16ml / h. During the induction period, the temperature is always maintained at 30℃, the speed is adjusted to 900rpm, the dissolved oxygen is controlled at 20%, and the pH is maintained at 6 by feeding with ammonia water; after about 48h of induction, the protein starts to degrade by SDS-PAGE detection or the protein concentration measured by UV does not show a significant increase, then the tank can be released.

[0135] The fermentation supernatant was collected and subjected to SDS-PAGE electrophoresis. Figure 3 As shown, under high-density fermentation conditions, at 72 h of induction, collagens JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, and JU-3-3 had almost only the target bands, and the main bands in their optical density analysis accounted for more than 95%.

[0136] 2. Recombinant collagen purification

[0137] The fermentation supernatant prepared in step 1 was collected and the cation exchange medium (chromatographic filler was SP from Suzhou Lanxiao) was balanced with buffer A. FF was loaded into Jiaxing Qianchun Biological QCXKC-50 / 30) until the UV215 absorbance and conductivity values ​​were stable, the sample flow rate was set to 35 mL / min, the sample volume was 1 L / time, and the UV215 absorbance was detected. When it rose, the sample was started.

[0138] After the sample loading is completed, continue to balance the cationic chromatography medium with buffer A. When the UV215 absorbance value drops to 400mAu, stop loading the sample and continue to flow until the UV and conductivity are balanced. Use 10% B and 20% B for elution respectively, collect the eluent, and detect the status of 10% B and 20% B purified proteins respectively. After determining the components, perform ultrafiltration desalting, followed by fine filtration, freeze drying, and collect freeze-dried collagen sponge. In addition to the purification method used in this example, the carboxyl end of collagen has a 6×His tag, and affinity media such as Ni-NTA and Ni-TED can be used for affinity purification, which can also achieve similar effects to this example.

[0139] The purified freeze-dried collagen sponge was dissolved in ultrapure water and subjected to SDS-PAGE electrophoresis. The SDS-PAGE electrophoresis results of JU-3-3 were as follows: Figure 4 As shown, the purity of HPLC was as Figure 5 As shown, the purity is >95%, and the freeze-dried recombinant collagen sponge is prepared as follows Figure 6 shown.

[0140] Example 3: Performance testing of recombinant collagen

[0141] The recombinant collagen freeze-dried collagen sponge prepared in Example 2 was taken to test its performance.

[0142] 1. Stability

[0143] The recombinant protein freeze-dried sponges (JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, JU-3-3) were added with ultrapure water to prepare a 10 mg / mL solution, filtered through a 0.22 μm filter membrane and dispensed into sterile centrifuge tubes. The solid protein sponges were heated at 60°C for 10 days and 121°C for 20 minutes, and samples were taken for SDS-PAGE detection.

[0144] The stability results of JU-3-3 are as follows Figure 7 As shown in A, the results show that the treated JU-3-3 still maintains structural and purity stability, with no significant difference from day 0; at the same time, treatment at 121°C for 20 minutes does not cause JU-3-3 degradation; the results of the competitor (mainstream collagen in the market) are as follows Figure 7 As shown in B, the results at 60°C and 121°C for 20 min indicate that the protein structure has been destroyed, and severe degradation and diffusion have occurred, proving that JU-3-3 has extremely high thermal stability.

[0145] The results of JU-4-2, JU-4-3, JU-4-6, JU-4-7, and JU-4-8 are as follows Figure 7 As shown in C, the results show that it remains stable at different temperatures, but JU-3-3 has better stability.

[0146] 2. Cell adhesion and proliferation activity

[0147] Normally culture NIH / 3T3 cells (purchased from the cell bank of the Chinese Academy of Sciences, catalog number GNM6, culture and passage methods refer to the cell manual). Take recombinant collagen freeze-dried sponges JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, JU-3-3, control bovine collagen (China National Institute for Food and Drug Control, catalog number 380002) and natural human collagen (sigma), and measure the concentration. Add 100 μL of various protein solutions and blank PBS solution to a 96-well cell culture plate and let it stand at room temperature for 60 minutes; then add 10 5 3T3 cells in good culture state were incubated at 37°C and 5% CO2 for 60 min; the cells in the wells were washed 4 times with PBS.

[0148] Refer to the recombinant collagen adhesion and proliferation detection method in the recombinant collagen industry standard YY1849-2022 for determination.

[0149] The higher the adhesion activity, the more collagen can help cells adhere to the wall or extracellular matrix in a short time, which can better build a cell growth microenvironment and better exert the effect of collagen. Figure 8 As shown, the results showed that the adhesion activity of JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, and JU-3-3 was close to that of bovine collagen, with no significant difference, and were all significantly higher than that of the competitor group.

[0150] The higher the proliferation activity, the more collagen can stimulate cell regeneration while providing a microenvironment, and the better the proliferation effect, indicating that the activity of collagen is strong. Fig. 9 As shown, the results showed that the adhesion activity of JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, and JU-3-3 was close to that of natural bovine collagen, and were significantly higher than that of the competitor group.

[0151] 3. Security

[0152] According to GB / T16886.5-2017, the test steps are as follows:

[0153] The potential cytotoxicity of recombinant collagen JU-3-3 was tested by in vitro culture of mammalian L-929 cells. Recombinant collagen (1 mg / mL, 4 mg / mL, 8 mg / mL) and control sample (10% DMSO) were placed in MEM culture medium containing 10% fetal bovine serum and extracted in a 37°C incubator for 24 hours. After the extraction, the 96-well plate (10 4 The cell culture medium in each well was removed and replaced with the corresponding extract, and the cells were cultured in a cell culture incubator (37° C., 5% CO 2 , >90% humidity) for 24 hours.

[0154] After the culture, the cell morphology and cell lysis were observed under a microscope, and the cytotoxicity value of the test sample was determined by the MTT method. Fig.10 As shown, the results showed that the blank control group and cells had normal morphology throughout the experiment and showed no cytotoxic reaction; the positive control group (10% DMSO) showed severe cytotoxic reaction; after incubating the cells at a concentration of 8 mg / mL for 24 hours, the cell morphology was basically intact and the cell viability value was 97.6%. It can be seen that the recombinant collagen JU-3-3 had no cytotoxic effect under the MTT cytotoxicity test conditions.

[0155] 4. Acid-base tolerance and freeze-thaw stability testing

[0156] The stability of JU-3-3 under different pH conditions was tested. Fig.11As shown in A in the figure, the results show that JU-3-3 has good pH applicability, maintains strong stability at pH 6-8, and has good clinical pH applicability.

[0157] Freeze-thaw stability results Fig.11 As shown in B, it can be seen that JU-3-3 also has good freeze-thaw stability (repeated freeze-thaw at -20°C and -80°C), which is convenient for storage and use.

[0158] Example 4: Application of recombinant collagen in the preparation of hydrogel

[0159] The recombinant collagen freeze-dried sponge JU-3-3 prepared in Example 2 was used to prepare a hydrogel in the following steps:

[0160] The collagen hydrogel was obtained by using PEG-NHS activated lipid as the cross-linking agent at a concentration of 5 mg / ml and collagen at 40 mg / mL at 37°C for 30 min.

[0161] The appearance of the gel before and after heat treatment is as follows Fig.12 As shown, the appearance of the gel did not change before and after heat treatment (121°C, 20 min); the dynamic viscosity was tested by rheometer and was 328.09 Pa / s and 306.58 Pa / s respectively, and the dynamic viscosity did not change significantly. Fig.13 The results show that collagen hydrogel has gelling properties, and the structure is intact at 121°C for 20 minutes, and can be sterilized by wet heat to ensure the sterility of the product and meet the requirements for medical device implantation.

[0162] Sequences used in the present invention:

[0163] COL3A1 amino acid sequence SEQ ID NO.1:

[0164]

[0165] JU-4-2 recombinant collagen monomer fragment 1 amino acid sequence SEQ ID NO.2:

[0166] GEKGETGAPGLKGENGLPGENGAPGPMGPRGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGAPGPQGPRGDK

[0167] JU-4-2 recombinant collagen single chain amino acid sequence SEQ ID NO.8:

[0168] GEKGETGAPGLKGENGLPGENGAPGPMGPRGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPRGEPGKNGAK GEPGPRGERGEAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPRGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGAPGPQGPRGDKGEK GETGAPGLKGENGLPGENGAPGPMGPRGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPRGEPGKNGAKGEP GPRGERGEAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPRGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGAPGPQGPRGDKHHHHHHH

[0169] JU-4-3 recombinant collagen monomer fragment 2 amino acid sequence SEQ ID NO.3:

[0170] GEKGETGAPGLKGENGLPGENGAPGPMGPKGDPGKNGAKGEPGPKGDKGDAGIPGVPGAKGEDGAPGPQGPRGDK

[0171] JU-4-3 recombinant collagen single chain amino acid sequence SEQ ID NO.9:

[0172] GEKGETGAPGLKGENGLPGENGAPGPMGPKGDPGKNGAKGEPGPKGDKGDAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPKGDPGKNGAKGEPGPKGDKGDAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPKGDPGKNGAKGEPGPKGDKGDAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPKGDPGKNGAKGEPGPKGDKGDAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPKGDPGKNGAKGEPGPKGDKGDAGIPGVPGAKGEDGAPGPQGPRGDKGEKGETGAPGLKGENGLPGENGAPGPMGPKGDPGKNGAKGEPGPKGDKGDAGIPGVPGAKGEDGAPGPQGPRGDKHHHHHH

[0173] Amino acid sequence of JU-4-6 recombinant collagen monomer fragment 3, SEQ ID NO.4:

[0174] GPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGAPGPQGPRGDK

[0175] Amino acid sequence of JU-4-6 recombinant collagen single chain, SEQ ID NO.10:

[0176] GPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPHHHHHHJU-4-7 recombinant collagen monomer fragment 4, amino acid sequence SEQ ID NO.5:

[0177] GEKGETGAPGLKGENGLPGENGAPGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGERGAAGIK

[0178] JU-4-7 recombinant collagen single chain SEQ ID NO.11:

[0179] GEKGETGAPGLKGENGLPGENGAPGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGERGAAGIKGEKGETGAPGLKGENGLPGENGAPGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGERGAAGIKGEKGETGAPGLKGENGLPGENGAPGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGETGERGAAGIKHHHHHH

[0180] JU-4-8 recombinant collagen monomer fragment 5 SEQ ID NO.6:

[0181] GPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGNTGAPGSPGVSGPKGDAGQPGEKGS PGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDK

[0182] JU-4-8 recombinant collagen single chain SEQ ID NO.12:

[0183] GPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGSPGAPGAPGHPGPPGPVGPAGKSGAPGPQGPRGDKGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPHHHHHH

[0184] Amino acid sequence of JU-3-3 recombinant collagen monomer fragment 6, SEQ ID NO.7:

[0185] GPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGE RGPPGERGSEGSPGHPGQPGPPGPPGAPGPQGPRGDK

[0186] Amino acid sequence of JU-3-3 recombinant collagen single chain, SEQ ID NO.13:

[0187] GPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGERGSEGSPGHPGQPGPPGPPGAPGPQGPRGDKGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGERGSEGSPGHPGQPGPPGPPGAPGPQGPRGDKGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGERGSEGSPGHPGQPGPPGPPGAPGPQGPRGDKGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGERGSEGSPGHPGQPGPPGPPGAPGPQGPRGDKHHHHHH

[0188] JU-4-2 Nucleotide sequence SEQ ID NO.14:

[0189] Ggtgaaaaaggtgaaacgggcgctccaggactcaagggagaaaatggtcttcccggtgagaacggagcacctggtccaatggggcctagaggcgaaccgggc

[0190] aaaaacggcgcgaagggcgagcccggcccgaggggtgaacgtggcgaggcggggattccgggagtgcctggtgctaaaggggaggatggtgcccccggacc

[0191] tcaggggccacgtggcgacaaaggggaaaagggagagactggtgccccgggtttaaaaggagagaacggcttacccggtgaaaatggtgctcctgggccaatg

[0192] ggaccccgcggtgaaccgggtaaaaatggtgcgaagggagaaccgggacctcgtggtgagcgaggagaggccggtattccgggggttcccggcgcaaagggg

[0193] gaggacggcgccccgggaccacaaggaccgcgaggcgacaagggggaaaagggcgagacaggagcgccggggttgaagggagagaatggccttcccggc

[0194] gagaacggtgctccaggaccaatgggcccccggggagaaccagggaagaacggcgcgaaaggggaacctggaccccggggagaaaggggggaggctggg

[0195] atacccggtgtaccgggggcaaaaggtgaggacggggcccccggaccacaagggccacgaggtgataaaggggaaaagggagaaaccggggcacctggcct

[0196] gaagggagaaaatgggctccctggcgaaaatggcgcgccgggaccaatggggccgaggggtgaacccggcaaaaatggtgcgaaaggagagccagggcctc

[0197] ggggtgaacgcggggaggcaggaatcccgggagtccctggtgctaagggtgaggatggcgcgcctggcccccaaggtccccgcggcgataaaggtgaaaaag

[0198] gggagacaggtgctcctggcctgaaaggtgaaaacggtctaccgggggagaacggtgcaccagggcccatggggcctaggggggaaccaggcaaaaatgggg

[0199] caaagggtgagccggggcctcgcggcgaaagaggtgaggcaggcataccaggcgtaccgggggcgaaaggggaagatggggcccccggaccgcagggtcc

[0200] tcggggcgacaaaggcgagaagggagaaactggggccccgggattgaagggtgagaacggactacccggcgagaacggagccccggggcctatgggtccaa

[0201] gaggcgaacctggcaagaatggggcaaagggagagccaggccctagaggagagcgtggagaggctggtatccccggggtgcccggcgccaaaggtgaagat

[0202] ggtgctcctggtcctcaggggccacgtggagacaag

[0203] Nucleotide sequence of JU-4-3: SEQ ID NO.15

[0204] Ggtgagaagggggagactggtgctcctgggcttaaaggtgaaaatggcctgcccggtgaaaatggcgcgcctggaccgatggggccaaagggggataagggg

[0205] gacccgggcaaaaacggggcaaaaggtgagccaggaccaaagggcgacaagggggatgcgggcattccaggagttccgggcgcgaaaggagaagatggag

[0206] cacctggcccccaagggcccaggggggacaagggagaaaagggggagacgggggcaccggggctgaaaggtgaaaacggtttacccggtgagaatggagc

[0207] gcctgggccaatgggaccgaagggagacaagggggacccagggaagaacggcgctaagggagagcccgggccaaagggagataaaggcgatgcagggata

[0208] cctggagtccccggtgctaagggagaagacggagccccaggtcctcagggcccacgtggcgataaaggggaaaaaggcgaaactggcgctccgggcctaaaa

[0209] ggcgagaacggactccccggtgaaaatggggcccctggaccgatgggaccaaaaggagacaaaggtgatcctgggaaaaatggagccaagggtgagccaggt

[0210] cctaaaggtgacaagggcgatgcaggaatccccggggttcccggtgccaaaggggaggatggcgcacccgggcctcaaggcccacggggagataaagggga

[0211] gaagggcgagaccggtgcacctggacttaagggtgaaaacggcttgcctggagagaatggcgcgcctggtccgatggggcccaagggcgacaaaggtgaccct

[0212] ggtaaaaacggggctaagggagaacctggtccgaaaggtgacaagggggacgccggaattccgggcgtgcctggtgcgaaaggcgaagacggtgcgcccggt

[0213] ccgcagggaccccgaggtgataaaggggagaagggcgagacaggcgcacccggattgaaaggcgaaaacggtttaccgggcgaaaatggagcccccggacc

[0214] catgggcccgaaaggagataagggtgaccctgggaaaaatggcgccaaaggtgaacctggtccaaaaggagataagggggacgctggtatcccgggggtgcct

[0215] ggcgccaaaggggaagacggtgctcccggaccccaaggcccgcgcggggataagggcgaaaagggtgagaccggcgctcctggtctcaagggagaaaacgg

[0216] gctaccgggggagaacggcgcgccagggccaatgggtccaaaaggcgataagggtgacccgggtaaaaatggtgcgaagggtgagccgggtcccaaagggg

[0217] The ataagggcgatgccggcataccgggagtaccgggagctaagggcgaggatggggcacccggaccacagggaccaagaggagacaaa JU-4-6 nucleotide sequence SEQ ID NO.16:

[0218] Ggaccgcccggtgagaatggtaaacccggtgagcctggaccgaaaggcgacgctggggcgccaggggctccaggcggtaaaggagacgccggcgcccccg

[0219] gggagcgtggaccgccaggtgctccagggccccagggacctaggggtgataagggccctccgggcgagaacgggaagccgggagaaccaggtccaaaggg

[0220] ggatgcgggggcacctggggcacccgggggtaaaggggacgcgggcgcacctggagagcgagggcccccgggggcgcctggtccacaaggcccgagagg

[0221] agacaaaggaccgcctggagagaatggcaaaccgggggagccagggcccaaaggagacgcaggagcacccggtgctcccggcgggaagggagacgcggg

[0222] agcacccggtgagcggggtccgccgggtgccccgggcccgcagggcccgaggggagacaaaggaccacccggagagaacggcaaacctggggaaccggg

[0223] ccccaagggcgatgcgggtgcccctggggctccaggggggaagggggatgccggcgctcctggggagcgaggtccgcccggtgccccaggaccacagggg

[0224] ccgcgcggtgataaaggccccccaggtgaaaatggcaagccaggcgaaccaggacctaagggcgacgcaggtgctcctggagccccagggggcaagggaga

[0225] tgccggggcacctggggaacgtggtcctcctggtgcgcccggtcctcaaggtccccgcggtgacaagggcccgccgggagaaaatggaaagcctggggaacct

[0226] ggccccaaaggcgacgcaggcgccccaggcgcacctgggggtaaaggagatgcgggggccccgggtgagagaggtcccccgggggctccgggtccccaag

[0227] gacctcgtggggataagggcccccctggggaaaacggaaagcctggcgaaccaggacctaaaggtgacgcgggggcgcccggcgcgccgggcggcaaggg

[0228] ggacgctggtgcccctggcgaaaggggccctccaggcgctccaggtccacaagggccccggggagataaaggtccaccgggagaaaacggaaaacctggcga

[0229] gccgggtcccaagggtgatgcaggagcacccggtgcgccgggaggtaaaggagatgctggggccccaggagaacgcggccccccaJU-4-7 nucleotide sequence SEQ ID NO.17:

[0230] Ggggagaagggcgagactggcgcgcctggtctaaagggcgagaatggacttcccggggaaaatggggctcccggcgagccggggaagaacggagcgaaag

[0231] gcgaacccggcccccgcggtgagcgaggagaggcgggtataccgggagtcccgggggctaaaggagaggacggaaatacaggagcgcctggtagtcccggg

[0232] gtgagtggtccaaaaggcgatgccggacaacctggcgagaagggaagccctggtgctcaaggacccccaggagcaccaggaagccccggcgcaccaggtgcc

[0233] cccggccaccctgggccaccgggaccggttggacccgcagggaagtctggcgctccgggaccccagggccccagaggagacaagggagagacaggagagc

[0234] gcggagccgcaggcattaagggcgaaaaaggtgagactggtgcaccagggctgaagggagaaaacgggttaccgggggagaatggagcaccaggcgaaccg

[0235] ggaaagaacggcgcgaaaggtgaaccgggtccacgtggggaacggggagaagccgggatacctggcgtacctggtgctaaaggagaagacggcaacaccgg

[0236] ggcacccgggtcccctggagtttcggggcccaaaggtgatgccgggcagccaggcgagaaagggtcaccgggtgcacaaggacccccgggtgcaccagggtc

[0237] tccaggagccccaggggcgccagggcatcctggtcctccagggccagtcggtcctgccggcaagtccggggctccgggcccccaaggtcccaggggtgacaaa

[0238] ggtgaaacgggggaacggggcgcggcaggcatcaagggggaaaagggagagacgggagcacctggcttgaagggggaaaacggtctccccggtgaaaatg

[0239] gcgctccgggggagccagggaaaaatggtgccaaaggggaacctggcccaaggggtgagcgtggtgaagcgggcatccctggggtgcctggcgcgaaagga

[0240] gaggacggcaacaccggtgcgcccggttctccgggcgtctccggcccaaaaggggatgctggccaaccaggtgaaaagggctcgcccggtgcccagggtccac

[0241] cgggtgctcctggttcgccgggagcgccgggtgcccctggccacccgggacctccgggtcccgtaggcccagccgggaaatcaggggctcctggtcctcagggt

[0242] cctcgtggtgataagggggaaaccggtgagagaggcgcggctggaattaaa

[0243] JU-4-8 nucleotide sequence SEQ ID NO.18:

[0244] ggaccacctggtgagaatggcaaaccgggtgagccggggccaaaaggggatgccggtgctcctggggcccccggtggtaagggtgacgccggcgcacctgga

[0245] gaacgggggccgcctggtaacaccggtgcacccggctcgccgggagtctccggaccgaaaggcgacgcaggtcagccaggtgagaagggtagtcccggggc

[0246] gcaggggccacccggtgctcccggaagcccgggtgcaccaggggctcctggccatcccggccctccaggccccgtaggccctgccgggaagtcgggagcccc

[0247] aggacctcaaggaccccggggtgataaaggaccaccaggagaaaacggcaaaccgggcgaaccaggaccgaagggggacgcgggggcaccaggtgctcct

[0248] ggtggaaaaggggacgccggtgctcctggcgagcgtggcccacctggtaacacgggagcaccagggtctcccggggtttccggtccaaagggggatgcgggac

[0249] aacccggagaaaaagggagtcccggtgctcaagggcctcccggagcacctggtagccccggggcgcctggcgcacctggacacccaggaccccctggaccag

[0250] ttgggccggcgggtaaaagcggcgcgccaggcccacagggccctagaggagacaaaggacccccgggagaaaatggcaaacctggagaaccgggaccgaa

[0251] gggcgatgcgggggctccaggggcccctgggggcaagggcgacgcaggtgcaccgggggaacgagggccgcccggcaacactggggcgccagggtcccct

[0252] ggcgtaagtggccctaaaggggatgcgggccagcctggcgagaaagggagcccaggggctcaaggcccgccgggagcacctgggtcgccgggcgcgccgg

[0253] gcgcccccggtcatcctgggccccctggacctgtgggtcccgcaggaaagtcaggggctcccggtccacaaggccctaggggcgataaggggcctccaggtga

[0254] aaatggaaaacccggcgagccagggcccaagggggacgccggggcgcctggtgcgccgggcgggaagggtgatgcaggtgctcctggtgaaagggggcca

[0255] cccggaaatacaggggccccaggttctccaggcgtctcaggtccgaagggagacgccggtcagcccggggagaaaggttcgccaggtgctcaaggtcctcctgg

[0256] agcacctggttctccgggggcgcccggtgcgcctggccaccctggtccaccagggccggtgggcccggcgggtaaatcaggcgctcccggaccccaaggaccg

[0257] cgtggcgacaaagggccccccggagaaaatggaaagccgggcgagccaggcccgaagggagatgccggcgctccgggtgctccgggtggtaagggagatgc

[0258] cggtgccccgggagagcgcggcccgccc

[0259] JU-3-3 nucleotide sequence SEQ ID NO.19:

[0260] Gggccaccaggcccaaccgggccaggtggagacaagggcgatacgggaccaccagggccacaaggtttgcaggggcttccaggtacgggtggccctccagg

[0261] ggaaaacggcaagccgggggaacctggaccgaaaggtgacgcgggtgctcccggtgcgcctggcggtaaaggcgatgctggggcgccgggggagcgagga

[0262] ccaccgggcgagcgaggctccgagggatcaccgggacatccgggtcagcccggaccgcccggcccacctggtgcccctggtcctcaaggcccacgtggcgata

[0263] aggggccacccggtcccacaggccccggaggtgataaaggtgatactggcccgccaggaccgcaaggcttacagggcctcccgggaactggggggcccccgg

[0264] gcgaaaacgggaaaccaggcgaaccaggtcctaaaggcgacgcgggcgcacctggcgctcctggggggaaaggagacgctggggctcccggagagagaggt

[0265] cctccgggcgaacgcggatcggagggcagtccggggcacccgggccaaccggggcctccgggtcctcccggagcaccaggtccccaaggaccccgtgggga

[0266] taagggacctccgggccccacaggaccaggtggcgacaagggtgataccgggcctcccggtccgcaaggactacagggcctgcccgggaccgggggaccccc

[0267] cggtgaaaatggaaaacccggagagcctgggccaaagggggacgcgggtgcaccaggtgcccctgggggaaaaggtgatgccggagcacccggggagagg

[0268] gggccccctggcgaaaggggctctgaaggatcaccgggacacccaggccagccggggccgcccggtcctccgggtgcacctgggccgcaaggacctcggggt

[0269] gacaagggaccgcccgggcccacaggccctggaggagacaagggtgatactggtccgccaggtcctcaggggctgcaaggactcccaggaacgggtgggcca

[0270] ccaggagagaatggtaagcctggcgaacctgggcctaagggagacgctggcgcccctggcgccccaggagggaaaggagacgcaggcgccccaggagaga

[0271] gaggaccgccaggcgaacgcgggtctgagggtagccccggccatcctgggcagcccgggccccccggtcccccgggcgcgccggggcctcagggtccccgg

[0272] ggtgataaa

[0273] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 recombinant collagen single chain, characterized in that: The collagen single chain contains repeated collagen monomer segments; The amino acid sequence of the collagen monomer fragment is shown in any one of SEQ ID NOs. 2 to 7.

2. The recombinant collagen single chain according to claim 1, characterized in that: The collagen single chain contains 2 to 10 collagen monomer fragments; Optionally, the collagen single chain contains 3 to 7 collagen monomer fragments; Optionally, the collagen single chain contains a collagen monomer fragment with 3 to 7 amino acid sequences as shown in SEQ ID NO.

7.

3. The recombinant collagen single chain according to any one of claims 1 to 2, characterized in that: The recombinant collagen single chain is obtained by cutting and splicing on the basis of human type III collagen; Optionally, the amino acid sequence of the human type III collagen is as shown in SEQ ID NO.

1.

4. A gene encoding the single-chain collagen according to any one of claims 1 to 3.

5. A plasmid or cell carrying a gene encoding the single-chain collagen according to any one of claims 1 to 3.

6. Use of the recombinant collagen single chain according to any one of claims 1 to 3, the gene according to claim 4, the plasmid or the cell according to claim 5 in the preparation of collagen products.

7. The use according to claim 6, characterized in that: The collagen products include food, health products, medicines, medical devices or cosmetics; Optionally, the food includes functional food and food additives; Optionally, the medicine includes a wound repair material, a cartilage repair material, and a drug delivery carrier; Optionally, the medical device includes tissue engineering materials, surgical auxiliary equipment, and oral repair materials; Optionally, the cosmetics include skin care products, hair care products, and anti-aging products.

8. A collagen product, characterized in that: The product contains the recombinant collagen single chain according to any one of claims 1 to 3; Optionally, the collagen product includes food, health care product, medicine, medical device or cosmetic.

9. A collagen hydrogel, characterized in that: The method is prepared by using the recombinant collagen single chain according to any one of claims 1 to 3, and the preparation method comprises: The recombinant collagen single chain and the cross-linking agent are mixed, and the collagen hydrogel is obtained by cross-linking reaction; Optionally, the cross-linking agent includes PEG-NHS activated lipid, EDC-NHS, glutaraldehyde; Optionally, the cross-linking reaction is carried out at 35-40° C. for 20-60 min; Optionally, the concentration ratio of the cross-linking agent to the recombinant collagen single chain is 1:20 to 1:

100.

10. A method for improving the performance of a recombinant collagen single chain, characterized in that: The performance includes one or more of stability, biological activity, and safety; The method comprises: Using repeated collagen monomer segments to form a single chain of recombinant collagen; Wherein, the amino acid sequence of the collagen monomer fragment is shown in any one of SEQ ID NOs. 2 to 7; Optionally, the collagen single chain contains 2 to 10 collagen monomer fragments; Optionally, the collagen single chain contains a collagen monomer fragment with 3 to 7 amino acid sequences as shown in SEQ ID NO.7.

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