Anti-degradation recombinant collagen, and preparation method and application thereof

Abstract

This invention discloses an anti-degradation recombinant collagen, its preparation method, and its applications, belonging to the fields of collagen and bioengineering technology. This invention selects the full-length or partial genes of natural human collagen, performs optimized combinations, and designs a collagen amino acid sequence with high stability and bioactivity. Theoretically, this amino acid sequence is completely consistent with the corresponding region sequence of natural human collagen, exhibits low immunogenicity, and possesses the typical biological functions of human collagen. The recombinant collagen prepared by this invention exhibits excellent stability, bioactivity, and safety.

Description

Technical Field

[0001] This invention relates to an anti-degradation recombinant collagen, its preparation method and application, belonging to the fields of collagen and bioengineering technology. Background Technology

[0002] Collagen, as a medical biomaterial, has demonstrated unique advantages in the medical field, the most prominent of which is its low immunogenicity. This characteristic significantly reduces the risk of immune rejection when collagen is applied to the human body, thus ensuring the safety and effectiveness of the treatment process. Furthermore, it shows broad application prospects as an implant material, tissue repair material, and drug carrier.

[0003] Collagen also possesses excellent biocompatibility. In its interaction with host cells and tissues, collagen can rapidly integrate into the surrounding matrix, providing strong support for cell growth and differentiation. Collagen also promotes intercellular communication and material exchange, thereby helping to maintain normal cellular physiological functions. This biocompatibility allows collagen to play a more stable and lasting role in medical applications.

[0004] In addition to its excellent biocompatibility and low immunogenicity, collagen possesses outstanding mechanical properties and structural stability, making it widely applicable in the field of biomechanics. Its high strength allows it to withstand various forms of external forces, including pressure and tension, without significant deformation or structural damage. This property makes collagen a key factor in maintaining the structural integrity and functional stability of organisms. Whether in skin, bone, or muscle tissues, collagen effectively resists the influence of the external environment, maintaining the morphological and functional stability of the tissues.

[0005] Existing patents for expressing recombinant collagen in yeast are mostly focused on expressing humanized collagen sequences, such as "Recombinant Human Collagen Fusion Peptide and Its Preparation Method and Application" (application number CN201010527766.7), "A Recombinant Human Type I Collagen and Its Immobilized Fermentation Production Method" (application number CN201510535383.7), and "Recombinant Human Type III Collagen α1 Chain and Its Application" (application number CN201911093124.8), which mostly involve the expression of collagen fragments or the fusion expression of different fragments. In addition, some patents involve the expression of artificially designed collagen-like sequences, such as "Human-like Collagen Gene, Its Different Number of Repeats in Tandem, Recombinant Plasmid Containing Tandem Gene and Preparation Method" (application number CN200610098297.5). The proteins expressed in these patents are all linear single-stranded, inevitably containing sites that are easily deamidated, easily hydrolyzed, or easily oxidized. Furthermore, while the human-like collagen sequence involved in patent CN200610098297.5 is artificially designed, the design concept is not explained, and the contribution of the artificially designed sequence to product stability is not clearly stated. "Highly Stable Recombinant Collagen, Construction Method and Its Application" (application number: CN202210954027.9) obtains recombinant collagen sequences with improved stability through amino acid sequence mutations, thereby improving protein yield and storage stability. Although this ensures high sample stability, it involves amino acid sequence mutations, posing a safety risk.

[0006] The aforementioned technologies involving recombinant collagen may introduce increased instability during solution preparation or use, potentially leading to process-related impurities and uncontrollable degradation products, increased toxicity of unknown substances, and increased immunogenicity. Furthermore, while some patents improve stability by mutating collagen amino acids, the mutated collagen sequence differs from the human collagen sequence, potentially leading to its recognition as a foreign substance during use, increasing immunogenicity risks, posing certain safety risks, and increasing other uncontrollable risks, thus increasing the risks associated with clinical application.

[0007] Therefore, there is an urgent need to develop a collagen based on natural human collagen, which is non-mutated in amino acids, safe, and has strong anti-degradation properties and bioactivity, and has extremely high practical and economic value. Summary of the Invention

[0008] To address the problems of collagen's easy degradation in vitro, increased immunogenicity, low biological activity, increased difficulty in purification and storage, and increased difficulty in quality control due to degradation during application, this invention selects the full-length or partial genes of natural collagen in the human body, performs certain optimized combinations, and designs a collagen amino acid sequence with strong stability and high biological activity. Theoretically, its amino acid sequence is completely consistent with the corresponding region sequence of natural human collagen, has low immunogenicity, and maintains the typical biological functions of human collagen.

[0009] The first objective of this invention is to provide a recombinant collagen single chain containing repeating collagen monomer fragments;

[0010] The amino acid sequence of the collagen monomer fragment is shown in any one of SEQ ID NO.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 a sequence of 3 to 7 amino acids 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 shown in SEQ ID NO.1.

[0016] In one embodiment, the recombinant collagen single chain is composed of collagen monomer fragments as shown in SEQ ID NO.2 repeated 2 to 10 times; wherein, the collagen monomer fragments as shown in SEQ ID NO.2 are obtained by sequentially combining 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);

[0017] Preferably, the procedure 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 collagen monomer fragments as shown in SEQ ID NO.3 repeated 2 to 10 times; wherein, the collagen monomer fragments as shown in SEQ ID NO.3 are obtained by sequentially combining 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 amino acid RGE with KGD;

[0019] Preferably, the procedure 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 collagen monomer fragments as shown in SEQ ID NO.4 repeated 2 to 10 times; wherein, the collagen monomer fragments as shown in SEQ ID NO.4 are obtained by sequentially combining 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 repetition is repeated 3 to 7 times; more preferably, it is repeated 6 to 8 times.

[0022] More preferably, in addition to repeating the sequence 6 to 8 times, amino acid sequences at positions 648 to 686 are additionally linked to the C-terminus of the sequence.

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

[0024] Preferably, the procedure 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 collagen monomer fragments as shown in SEQ ID NO.6 repeated 2 to 10 times; wherein, the collagen monomer fragments as shown in SEQ ID NO.6 are obtained by sequentially combining amino acids 648 to 686, 909 to 944, 1038 to 1061, and 648 to 686 of human type III collagen (amino acid sequence as shown in SEQ ID NO.1);

[0026] Preferably, the repetition is repeated 3 to 7 times; more preferably, it is repeated 3 to 5 times.

[0027] More preferably, in addition to repeating the sequence 3 to 5 times, amino acid sequences at positions 648 to 686 are additionally linked to the C-terminus of the sequence.

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

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

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

[0031] A 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 plasmids or cells carrying any of the above-mentioned collagen single-chain genes.

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

[0034] Optionally, plasmids include pET-28a, pET-32a, pGEX-4T-1, pBAD / Myc-His, and 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, plasmids include pcDNA3.1, pCMV-Tag, pCMV-Sport, pIRES2-EGFP, pEGFP-C1, pYES2, pPICZ, and 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; and mammalian cells such as HEK293 cells and CHO cells.

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

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

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

[0041] Optionally, the drug includes wound repair materials, cartilage repair materials, and drug delivery carriers;

[0042] Optionally, the medical device includes tissue engineering materials, surgical aids, and dental prosthetic materials;

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

[0044] In one embodiment, functional foods include, but are not limited to, collagen peptide drinks, such as collagen fruit juice drinks and collagen-containing coffee; and collagen candies, such as collagen compressed candies and collagen gummies.

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

[0046] In one embodiment, health 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 healing materials, such as collagen sponge (to promote wound healing), collagen hemostatic dressing (for rapid hemostasis), and collagen membrane (for postoperative repair).

[0048] Cartilage repair materials, such as collagen injections (for repairing articular cartilage defects) and collagen-based cartilage grafts;

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

[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 filler materials (for filling tissue or filling cavities).

[0051] Surgical aids include, but are not limited to, collagen sutures (biodegradable sutures) and collagen hemostatic sponges (for 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, cosmetics include skincare products, such as collagen masks (moisturizing, repairing, and anti-aging), collagen serums (improving skin elasticity), and collagen lotions (hydrating and locking in moisture).

[0054] Hair care products, such as collagen shampoo (to repair damaged hair) and collagen conditioner (to replenish nutrients and make hair smooth);

[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] A fourth object of the present invention is to provide a collagen product containing any of the above-described recombinant collagen single chains;

[0057] Optionally, the collagen product may include food, health products, pharmaceuticals, medical devices, or cosmetics.

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

[0059] Optionally, the drug includes wound repair materials, cartilage repair materials, and drug delivery carriers;

[0060] Optionally, the medical device includes tissue engineering materials, surgical aids, and dental prosthetic materials;

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

[0062] In one embodiment, functional foods include, but are not limited to, collagen peptide drinks, such as collagen fruit juice drinks and collagen-containing coffee; and collagen candies, such as collagen compressed candies and collagen gummies.

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

[0064] In one embodiment, health 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 healing materials, such as collagen sponge (to promote wound healing), collagen hemostatic dressing (for rapid hemostasis), and collagen membrane (for postoperative repair).

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

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

[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 filler materials (for filling tissue or filling cavities).

[0069] Surgical aids include, but are not limited to, collagen sutures (biodegradable sutures) and collagen hemostatic sponges (for 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, cosmetics include skincare products, such as collagen masks (moisturizing, repairing, and anti-aging), collagen serums (improving skin elasticity), and collagen lotions (hydrating and locking in moisture).

[0072] Hair care products, such as collagen shampoo (to repair damaged hair) and collagen conditioner (to replenish nutrients and make hair smooth);

[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] This 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 includes:

[0075] Recombinant collagen single chains and cross-linking agents are mixed, and a cross-linking reaction is carried out to obtain collagen hydrogel;

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

[0077] Optionally, the crosslinking 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] A fifth objective of this invention is to provide a method for improving the performance of recombinant collagen single chains, wherein the performance includes one or more of stability, bioactivity, and safety.

[0080] The method includes:

[0081] Recombinant collagen single chains are composed of repeated collagen monomer fragments;

[0082] The amino acid sequence of the collagen monomer fragment is shown in any one of SEQ ID NO.2 to 7;

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

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

[0085] Beneficial effects of the present invention

[0086] This invention selects the full-length or partial genes 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 collagen in the human body, has low immunogenicity, and has the typical biological functions of human collagen.

[0087] Specifically,

[0088] (1) The recombinant collagen prepared by this invention has excellent stability. It maintains structural and purity stability after being heated at 60°C for 10 days and at 121°C for 20 minutes. It also has good acid and alkali (pH 6-8) tolerance and freeze-thaw (-20°C, -80°C, three cycles) stability.

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

[0090] (3) The recombinant collagen prepared by this invention has basically intact cell morphology after 24 hours of cell incubation, with a cell viability of 97.6%, no cytotoxicity, and high biocompatibility;

[0091] (4) The hydrogel prepared by the recombinant collagen based on the present invention does not change in appearance before and after heat treatment (121℃, 20min) and has little effect on dynamic viscosity. Attached Figure Description

[0092] Figure 1 This is a schematic diagram of the recombinant plasmid.

[0093] Figure 2 SDS-PAGE analysis results of the supernatant from recombinant collagen expression in Pichia pastoris genetically engineered bacteria;

[0094] Figure 3 The results of SDS-PAGE analysis of the supernatant from large-scale fermentation are shown.

[0095] Figure 4 The results of SDS-PAGE analysis of recombinant collagen after purification and lyophilization;

[0096] Figure 5 The results are obtained by HPLC analysis after purification and lyophilization of recombinant collagen.

[0097] Figure 6 Appearance of purified lyophilized recombinant collagen samples;

[0098] Figure 7 The results are for the stability testing of recombinant collagen; where A represents the thermal stability of recombinant collagen single chain JU-3-3; B represents the thermal stability of competing products; and C represents the thermal stability of JU-4-3, JU-4-6, JU-4-7, and JU-4-8.

[0099] Figure 8 Results of recombinant collagen cell adhesion activity assay;

[0100] Figure 9 Results of recombinant collagen cell proliferation activity assay;

[0101] Figure 10 The results are from the recombinant collagen cytotoxicity assay.

[0102] Figure 11 Results of acid-base tolerance and freeze-thaw stability tests for recombinant collagen;

[0103] Figure 12 The appearance and structure of recombinant collagen hydrogel before and after heat treatment;

[0104] Figure 13 The dynamic viscosity of the recombinant collagen hydrogel before and after heat treatment. Detailed Implementation

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

[0106] Raw materials used in the examples:

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

[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); fed medium (50% w / v glycerol, with 12 ml PTM1 trace element per liter);

[0109] Induction medium (100% methanol, with 12 mL of PTM1 trace elements added per liter); PTM1: sterilized by filtration through a 0.22 μm filter membrane and stored at 4°C. After the fermentation medium is sterilized at high temperature, PTM1 is added after the temperature drops to room temperature, and the pH is adjusted to 4 with ammonia.

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

[0111] Buffer B: 25mM C6H8O7-Na3C6H5O7 (citric acid-sodium citrate) + 1M 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 sequences

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

[0117] (1) Take amino acids from positions 274 to 302, 432 to 464 and 1083 to 1095 in sequence to obtain recombinant collagen monomer fragment 1. The amino acid sequence is shown in SEQ ID NO.2. Repeat SEQ ID NO.2 6 times and attach a 6His tag to the C end to obtain recombinant collagen single chain, named JU-4-2. The amino acid sequence is shown in SEQ ID NO.8.

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

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

[0120] (4) Take amino acids from positions 273 to 296, 432 to 464, 909 to 944, 1038 to 1061 and 1083 to 1106 in sequence to obtain recombinant collagen monomer fragment 4. The amino acid sequence is shown in SEQ ID NO.5. Repeat SEQ ID NO.5 3 times and attach a 6His tag to the C-terminus to obtain a recombinant collagen single chain, named JU-4-7. The amino acid sequence is shown in SEQ ID NO.11.

[0121] (5) Take amino acid combinations from positions 648 to 686, 909 to 944, 1038 to 1061, and 648 to 686 in sequence to obtain recombinant collagen monomer fragment 5. The amino acid sequence is shown in SEQ ID NO. 6. Repeat SEQ ID NO. 6 4 times and connect the sequence from position 648 to 686 and the 6His tag to the C-terminus to obtain a recombinant collagen single chain, named JU-4-8. The amino acid sequence is shown in SEQ ID NO. 12.

[0122] (6) Take amino acid combinations from position 618 to 686, position 1170 to 1190, and position 1083 to 1095 in sequence to obtain recombinant collagen monomer fragment 6. The amino acid sequence is shown in SEQ ID NO.7. Repeat SEQ ID NO.7 4 times and attach a 6His tag to the C-terminus to obtain recombinant collagen single chain, named JU-3-3. The amino acid sequence is shown in SEQ ID NO.13.

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

[0124] The amino acid sequences SEQ ID NO. 8–13 obtained in step 1 were used to complete the corresponding nucleotide sequences (excluding the 6His tag, corresponding to nucleotide sequences SEQ ID NO. 14–19) by Nanjing Genscript Biotech Co., Ltd. These sequences were then inserted into the α-factor reading frame of the pPICzα empty vector (purchased from Thermo Fisher Scientific) to obtain recombinant plasmids, named pPICzα-JU-4-2 (amino acid sequence SEQ ID NO. 8, nucleotide sequence SEQ ID NO. 14), pPICzα-JU-4-3 (expressed amino acid sequence SEQ ID NO. 9, nucleotide sequence SEQ ID NO. 15), pPICzα-JU-4-6 (expressed amino acid sequence SEQ ID NO. 10, nucleotide sequence SEQ ID NO. 16), pPICzα-JU-4-7 (expressed amino acid sequence SEQ ID NO. 11, nucleotide sequence SEQ ID NO. 17), and pPICzα-JU-4-8 (expressed amino acid sequence SEQ ID NO. 12, nucleotide sequence SEQ ID NO. 19). The plasmids NO.18 and pPICzα-JU-3-3 (expressing amino acid sequence SEQ ID NO.13, nucleotide sequence SEQ ID NO.19) are illustrated in the diagram below. Figure 1 As shown.

[0125] The recombinant plasmid (3 μg) was digested with SacⅠ (purchased from Thermo Fisher) at 37℃ for 2 h, linearized, recovered, and electroporated into Pichia pastoris GS115 competent cells. The electroporated bacterial culture was plated on YPD / Zeocin plates and incubated upside down at 30℃ for 2–5 days. 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 pastoris genetically engineered strain obtained in step 2 was inoculated into 50 mL centrifuge tubes containing 5 mL of BMGY medium and cultured at 28–30 °C and 220 rpm until OD500. 600 The result was 10 (48 h). The cells were centrifuged at 3000 g for 5 min at room temperature, collected, and resuspended in BMMY medium to adjust the OD value. 600The culture medium was 10, and the culture was placed on a shaker at 30℃ and 220rpm for 3 days. Every 24 hours, 100% methanol was added to the culture medium until the final concentration was 1.0%. Bacterial samples were collected at 48h and 72h after methanol induction. The sample volume was 1mL and placed in a 1.5mL EP tube. The tube was centrifuged at 12000g for 5min at 4℃ and the expression supernatant was collected. The samples to be tested were stored at -80℃ for later use.

[0128] SDS-PAGE test results of supernatant Figure 2 As shown, the results indicate that all six collagen sequences can be secreted into the culture medium supernatant.

[0129] Example 2: Fermentation preparation of recombinant collagen

[0130] 1. Fermentation preparation of recombinant collagen

[0131] The Pichia pastoris genetically engineered strains 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. A fed-batch culture method was used, and the culture temperature was 30°C. The steps are as follows:

[0132] (1) Pichia pastoris genetic engineering was inoculated into 1L shake flasks containing 200mL of seed culture medium YPG and cultured at 220rpm and 30℃ for 22-24h until OD was reached. 600 Reaching 8-12;

[0133] (2) Use a 5L fermenter, fill with 2L of fermentation medium, adjust the rotation speed to 600rpm, the aeration rate to 3L / min, and the temperature to 30℃ before inoculation. Adjust the pH with the alkaline solution prepared with concentrated ammonia water and set the pH to 4. Inoculate 200mL of the prepared seed liquid into the tank (inoculate with a flame ring), click the dissolved oxygen electrode to calibrate to 100%, and start fermentation after calibration.

[0134] (3) When the dissolved oxygen level drops to 30% for the first time during growth, use the dissolved oxygen cascade rotation function to maintain it at 30%; wait for the glycerol to be depleted, the dissolved oxygen to rebound, and the dissolved oxygen level to be greater than 60% (OD). 600(Value approximately 50), begin glycerol feeding at a set rate of 40 mL / h. Stop feeding after approximately 250 mL of glycerol has been added and wait for dissolved oxygen to rise above 60%. Then, initiate methanol induction at a set feeding rate of 6 mL / h, increasing to 10 mL / h after two hours, and finally to 16 mL / h. During induction, maintain the temperature at 30°C, adjust the rotation speed to 900 rpm, control dissolved oxygen at 20%, and maintain pH 6 by adding ammonia. After approximately 48 hours of induction, once SDS-PAGE analysis indicates protein degradation or UV measurement shows no significant increase in protein concentration, the vessel can be removed from the tank.

[0135] Collect the fermentation supernatant and perform SDS-PAGE electrophoresis to detect the results. Figure 3 As shown, under high-density fermentation conditions, after 72 hours of induction, collagen JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, and JU-3-3 showed almost only the target bands, and the proportion of the main bands in the optical density analysis all exceeded 95%.

[0136] 2. Purification of recombinant collagen

[0137] Collect the fermentation supernatant obtained in step 1, and equilibrate the cation exchange medium (chromatographic packing material is SP produced by Suzhou Lanxiao) with buffer A. FF, loaded into Jiaxing Qianchun Bio QCXKC-50 / 30) until the UV215 absorbance and conductivity values ​​stabilized, the sample loading flow rate was set to 35 mL / min, and the sample loading volume was 1 L / time. The UV215 absorbance value was measured, and when it rose, the sample was started.

[0138] After sample loading, the cationic chromatography medium was equilibrated with buffer A. When the UV215 absorbance dropped to 400 mAu, sample loading was stopped, and the mixture was continued until UV and conductivity equilibrium was reached. Elution was performed with 10% B and 20% B, and the eluents were collected. The protein purification status of 10% B and 20% B was analyzed to determine the components. Ultrafiltration desalting was then performed, followed by purification filtration, freeze-drying, and collection of the lyophilized collagen sponge. Besides the purification method used in this example, collagen with a 6×His tag at the carboxyl terminus can also be purified using affinity media such as Ni-NTA and Ni-TED, achieving similar results.

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

[0140] Example 3: Performance testing of recombinant collagen

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

[0142] 1. Stability

[0143] Recombinant protein lyophilized sponges (JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, JU-3-3) were prepared into 10 mg / mL solutions with ultrapure water. After filtration through a 0.22 μm filter membrane, the solutions were aliquoted into sterile centrifuge tubes. Solid protein sponges were also heated at 60 °C for 10 days and treated at 121 °C for 20 minutes. Samples were then taken for SDS-PAGE analysis.

[0144] The stability results of JU-3-3 are as follows: Figure 7 As shown in A, the results indicate that the treated JU-3-3 maintained structural and purity stability, with no significant difference from day 0; simultaneously, treatment at 121℃ for 20 minutes did not cause JU-3-3 degradation; the results of competing products (mainstream collagen on the market) are as follows. Figure 7 As shown in B, the results indicate that the protein structure was destroyed and severely degraded and dispersed at 60℃ and 121℃ for 20 minutes, demonstrating that JU-3-3 has extremely high thermal stability.

[0145] The results for 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 indicate that the material remains stable at different temperatures, but JU-3-3 exhibits superior stability.

[0146] 2. Cell adhesion and proliferation activity

[0147] NIH / 3T3 cells were cultured normally (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GMN6; culture and passage methods were performed according to the cell instructions). Recombinant collagen lyophilized sponges JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, JU-3-3, control bovine collagen (National Institutes for Food and Drug Control, catalog number 380002), and natural human collagen (Sigma) were added to 96-well cell culture plates at a concentration of 2 mg / mL. 100 μL of each protein solution and blank PBS solution were added to each well, and the plate was incubated at room temperature for 60 min. Then, 10 μL of each protein solution was added to each well. 5 3T3 cells in good culture condition were incubated at 37°C and 5% CO2 for 60 min; the cells in the wells were washed 4 times with PBS.

[0148] The determination of recombinant collagen adhesion and proliferation was carried out in accordance with the YY1849-2022 industry standard for recombinant collagen.

[0149] The higher the adhesion activity, the faster collagen can help cells adhere to the cell wall or extracellular matrix, thus better constructing the cell growth microenvironment and maximizing the effects of collagen. Results are as follows: Figure 8 As shown, the results indicate that JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, and JU-3-3 have similar adhesion activities to bovine collagen with no significant differences, and all of them are significantly higher than the competing products.

[0150] Higher proliferation activity indicates that collagen, while providing a microenvironment, can also stimulate cell regeneration, resulting in better proliferation and thus stronger collagen activity. The results are as follows: Figure 9 As shown, the results indicate that JU-4-2, JU-4-3, JU-4-6, JU-4-7, JU-4-8, and JU-3-3 have similar adhesion activities to natural bovine collagen, and are all significantly higher than the competing products.

[0151] 3. Safety

[0152] The testing should be conducted according to GB / T16886.5-2017, and the steps are as follows:

[0153] The potential cytotoxicity of recombinant collagen JU-3-3 was tested using mammalian L-929 cells cultured in vitro. Recombinant collagen (1 mg / mL, 4 mg / mL, 8 mg / mL) and a control sample (10% DMSO) were placed in MEM medium containing 10% fetal bovine serum and incubated at 37°C for 24 hours. After extraction, 96-well plates (10... 4 Remove the culture medium from each cell (well), replace it with the appropriate extraction solution, and incubate in a cell culture incubator (37°C, 5% CO2, >90% humidity) for 24 hours.

[0154] After culture, cell morphology and cell lysis were observed under a microscope, and the cytotoxicity value of the test sample was determined using the MTT assay. Results are as follows: Figure 10 As shown, the results indicate that the blank control group and cells maintained 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 remained basically intact and the cell viability value was 97.6%. It can be seen that recombinant collagen JU-3-3 did not have any cytotoxic effect under the conditions of the MTT assay.

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

[0156] The stability of JU-3-3 under different pH conditions was tested, and the results are as follows: Figure 11As shown in A, the results indicate that JU-3-3 has good pH suitability, maintaining strong stability at pH 6–8, and has excellent clinical pH suitability.

[0157] Freeze-thaw stability results are as follows Figure 11 As shown in B, JU-3-3 also has good freeze-thaw stability (repeated freeze-thaw at -20℃ and -80℃), making it easy to store and use.

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

[0159] Take the recombinant collagen freeze-dried sponge JU-3-3 prepared in Example 2 and prepare a hydrogel, the steps are as follows:

[0160] A collagen hydrogel was obtained by crosslinking with PEG-NHS activated lipids at a concentration of 5 mg / mL and collagen at a concentration of 40 mg / mL at 37℃ for 30 min.

[0161] The appearance of the gel before and after heat treatment is as follows: Figure 12 As shown, the appearance of the gel remained unchanged before and after heat treatment (121℃, 20min); the dynamic viscosity measured by rheometer was 328.09 Pa / s and 306.58 Pa / s, respectively, indicating no significant change in dynamic viscosity. Figure 13 As shown in the figure. The results indicate that the collagen hydrogel possesses gel-forming properties and maintains its structural integrity at 121℃ for 20 min, enabling it to undergo moist heat sterilization, ensuring the product's sterility and meeting the requirements for medical device implantation.

[0162] The sequences used in this invention:

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

[0164]

[0165] The amino acid sequence of the first amino acid fragment of JU-4-2 recombinant collagen monomer is 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] The 2-amino acid sequence of the JU-4-3 recombinant collagen monomer fragment is 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] 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] Nucleotide sequence of JU-4-8: 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[[ID=​​

[0265] cctccgggcgaacgcggatcggagggcagtccggggcacccgggccaaccggggcctccgggtcctcccggagcaccaggtccccaaggaccccgtgggga

[0266] taagggacctccgggccccacaggaccaggtggcgacaagggtgataccgggcctcccggtccgcaaggactacagggcctgcccgggaccgggggaccccc

[0267] cggtgaaaatggaaaacccggagagcctgggccaaagggggacgcgggtgcaccaggtgcccctgggggaaaaggtgatgccggagcacccggggagagg

[0268] gggccccctggcgaaaggggctctgaaggatcaccgggacacccaggccagccggggccgcccggtcctccgggtgcacctgggccgcaaggacctcggggt

[0269] gacaagggaccgcccgggcccacaggccctggaggagacaagggtgatactggtccgccaggtcctcaggggctgcaaggactcccaggaacgggtgggcca

[0270] ccaggagagaatggtaagcctggcgaacctgggcctaagggagacgctggcgcccctggcgccccaggagggaaaggagacgcaggcgccccaggagaga

[0271] gaggaccgccaggcgaacgcgggtctgagggtagccccggccatcctgggcagcccgggccccccggtcccccgggcgcgccggggcctcagggtccccgg

[0272] ggtgataaa

[0273] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant collagen single chain, characterized in that, The amino acid sequence of the recombinant collagen single chain is shown in SEQ ID NO. 8~13.

2. A gene encoding the collagen single strand of claim 1.

3. A plasmid or cell carrying the single-stranded collagen gene of claim 1.

4. The use of the recombinant collagen single chain of claim 1, the gene of claim 2, the plasmid or cell of claim 3 in the preparation of collagen products.

5. The application according to claim 4, characterized in that, The collagen products include food, health products, pharmaceuticals, medical devices, or cosmetics.

6. The application according to claim 5, wherein the food includes functional foods and food additives.

7. The application according to claim 5, wherein the pharmaceutical product comprises wound repair material, cartilage repair material, and drug delivery carrier.

8. The application according to claim 5, wherein the medical device includes tissue engineering materials, surgical aids, and dental prosthetic materials.

9. The application according to claim 5, wherein the cosmetic includes skin care products, hair care products, and anti-aging products.

10. A collagen product, characterized in that, The product contains the recombinant collagen single chain as described in claim 1.

11. The collagen product according to claim 10, characterized in that, The collagen products include food, health products, pharmaceuticals, medical devices, or cosmetics.

12. A collagen hydrogel, characterized in that, The recombinant collagen single chain described in claim 1 is used to prepare the collagen using a preparation method comprising: Recombinant collagen single chains and cross-linking agents are mixed, and a cross-linking reaction is carried out to obtain collagen hydrogel; The crosslinking agent includes PEG-NHS activated lipid, EDC-NHS, and glutaraldehyde; The cross-linking reaction is carried out at 35~40℃ for 20~60 min; The concentration ratio of cross-linking agent to recombinant collagen single chain is 1:20 to 1:

100.

13. A method for improving the performance of recombinant collagen single chains, characterized in that, The performance includes one or more of stability, bioactivity, and safety; The method includes: Recombinant collagen single chains are composed of repeated collagen monomer fragments; The amino acid sequence of the recombinant collagen single chain is shown in SEQ ID NO.13.

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