A recombinant collagen and its application

Abstract

The present invention relates to the field of bioengineering and biomedical materials, and specifically to a recombinant collagen and its application. The present invention uses genetic engineering technology, utilizes the amino acid sequence of human type III collagen, optimizes and integrates the sequence on the basis thereof, and constructs a recombinant collagen, whose amino acid sequence is shown in SEQ ID No.1, or as shown in SEQ ID No.2. The recombinant collagen has better biocompatibility and biosafety, and is an innovative product with potential application prospects. The present invention fills the problems of seed source limitation, immunogenicity and infectious disease risk of animal-derived collagen, and provides a new direction for the development of collagen products. At the same time, the recombinant collagen can be used to prepare skin wound healing products, which has important application value and broad market prospects.

Description

Technical Field

[0001] The invention relates to the fields of bioengineering and biomedical materials, and in particular to a recombinant collagen and an application thereof. Background Art

[0002] Collagen is the most abundant protein in animals. It is an important structural protein that is widely distributed in the human body and is a key component for maintaining tissue structure and function. The distribution of human type III collagen in the human body is similar to that of type I collagen. It is widely present in tissues such as skin, bones, blood vessels and muscles, and is an important factor in maintaining tissue elasticity and strength. It is precisely because of the unique properties of collagen itself that it is widely used in medical, cosmetics and food fields.

[0003] However, traditional collagen products are mostly animal-derived, mainly from the skin and bones of animals such as cows and pigs, and there are some problems that limit their application. First, there are species restrictions on animal-derived collagen. The collagen composition and structure of different animals may vary, resulting in unstable product quality and performance. Secondly, animal-derived collagen has certain immunogenicity and may cause an immune response in the human body, leading to allergic or rejection reactions. In addition, animal-derived collagen also has infectious disease risks, such as the possibility of carrying viruses or bacteria, posing a potential threat to human health.

[0004] In order to overcome these problems, new recombinant collagen based on human collagen has become a research hotspot. In this context, the present invention provides a new recombinant collagen based on human type III collagen and its preparation method and application, in order to provide a new direction for the development of collagen products. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant collagen, a nucleic acid molecule encoding the recombinant collagen, a recombinant expression vector and a recombinant strain in view of the deficiencies in the prior art.

[0006] The technical problem that the present invention also aims to solve is to provide the use of the above recombinant collagen in the preparation of a product for healing skin wounds.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A recombinant collagen protein is constructed based on human type III collagen protein, and its amino acid sequence is shown as SEQ ID No.1 or as shown as SEQ ID No.2.

[0009] Preferably, the amino acid sequence of the recombinant collagen is as shown in SEQ ID No.2.

[0010] The nucleic acid molecule encoding the recombinant collagen, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 5 to 6.

[0011] Preferably, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.6.

[0012] A recombinant expression vector contains the nucleotide sequence of the nucleic acid molecule.

[0013] Specifically, the recombinant expression vector is pPIC9K-NOP1 or pPIC9K-NOP2, preferably pPIC9K-NOP2.

[0014] A recombinant strain uses Pichia pastoris as a host and contains the recombinant expression vector.

[0015] Specifically, the Pichia pastoris is Pichia pastoris GS115.

[0016] The use of the above-mentioned recombinant collagen, or the nucleic acid molecule encoding the recombinant collagen, or the recombinant expression vector, or the recombinant strain in the preparation of a skin wound healing product is also within the scope of protection of the present invention.

[0017] Wherein, the skin wound healing product is a hydrogel.

[0018] Specifically, the hydrogel is obtained by adding a recombinant collagen solution into a cellulose nanocrystal dispersion, mixing the mixture, and placing the mixture in a hydrochloric acid steam bath to stand.

[0019] Specifically, the concentration of the recombinant collagen solution is 40-80 g / L, and the solvent is 10-30 mM phosphate buffer solution with a pH of 4.49-6.80.

[0020] Preferably, the concentration of the recombinant collagen solution is 60 g / L, and the solvent is 20 mM phosphate buffer with a pH of 5.59.

[0021] Specifically, the cellulose nanocrystal dispersion is prepared by adding cellulose nanocrystals as a supporting skeleton into pure water at a mass volume ratio of 0.2 to 1.0%, and then subjecting the mixture to ultrasonic oscillation.

[0022] Preferably, the cellulose nanocrystals are added into pure water at a mass volume ratio of 0.6%, and then subjected to ultrasonic oscillation to obtain the cellulose nanocrystals.

[0023] Specifically, the recombinant collagen solution is added to the cellulose nanocrystal dispersion at a flow rate of 2 to 10 mL / min.

[0024] Preferably, the flow rate is 5 mL / min.

[0025] Specifically, the volume ratio of the recombinant collagen solution to the cellulose nanocrystal dispersion is 2:1 to 1:3.

[0026] Preferably, the volume ratio of the recombinant collagen solution to the cellulose nanocrystal dispersion is 1:1.

[0027] Specifically, the mixture is stirred and mixed at a rotation speed of 50 to 150 rpm.

[0028] Specifically, the standing time is 6 to 18 hours.

[0029] Preferably, the standing time is 12 hours.

[0030] Beneficial effects:

[0031] (1) The present invention uses genetic engineering technology to optimize and integrate the amino acid sequence of human type III collagen to construct two new types of recombinant collagen. The recombinant collagen provided is designed based on human type III collagen, ensuring a high degree of similarity with human type III collagen, and has obvious advantages in biocompatibility and biosafety, and can avoid the source restrictions, immunogenicity and infectious disease risks of animal-derived collagen.

[0032] (2) The two novel recombinant collagens designed by the present invention, especially the amino acid sequence shown in SEQ ID No. 2, are compared with natural type III human collagen. By optimizing and improving the amino acid residues in specific positions, the three-dimensional spatial structure and surface properties of the protein are changed. The hydrophilicity and stability of the optimized recombinant collagen are improved, thereby accelerating the wound healing process.

[0033] (3) The nucleic acid molecules of the present invention are optimized for the corresponding amino acid codons according to the codon preference of Pichia pastoris for expressing heterologous proteins. Pichia pastoris is used as the expression host. The present invention utilizes the efficient expression system of Pichia pastoris, which not only increases the yield but also reduces the production cost, making the recombinant collagen of the present invention more suitable for large-scale production.

[0034] (4) The composite hydrogel prepared from recombinant collagen and cellulose nanocrystals of the present invention has been shown to significantly promote wound closure and reduce scar formation in animal model skin wound healing experiments. Therefore, the present invention provides a new biomaterial with broad prospects for skin wound healing treatment, which can be used in the fields of medicine, biomaterials, and medical aesthetics, and has important application value and broad market prospects.

[0035] (5) The recombinant collagen and cellulose nanocrystals of the present invention construct a composite hydrogel, with cellulose nanocrystals as the supporting skeleton and hydrogen ions provided by a hydrochloric acid steam bath, so that the cellulose nanocrystals and the recombinant collagen form a multiple hydrogen bond network, thereby enhancing the mechanical strength and mechanical properties of the composite hydrogel peptide. In addition, no chemical cross-linking agents or toxic reagents are introduced during the preparation process, and the reaction conditions are mild and convenient. The prepared composite hydrogel has good biosafety and biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0037] Figure 1 Schematic diagram of the three-dimensional structure of recombinant collagen NOP2.

[0038] Figure 2 This is the recombinant plasmid map of recombinant collagen NOP1 and NOP2.

[0039] Figure 3 This is the SDS-PAGE electrophoresis diagram of recombinant collagen NOP1 and NOP2.

[0040] Figure 4 This is a picture of cellulose nanocrystal-recombinant collagen composite hydrogel (CNC-NOP1 hydrogel, CNC-NOP2 hydrogel).

[0041] Figure 5 This is a comparison chart of the mechanical properties of cellulose nanocrystal-recombinant collagen composite hydrogel.

[0042] Figure 6 This is a picture of wound healing and wound surface in mice.

[0043] Figure 7 This is a graph showing the wound healing rate in mice. DETAILED DESCRIPTION

[0044] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0045] This description is considered sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0046] In the following examples, the MD selection plate and BMMY medium are common mature formulas and can be found in the Pichia pastoris manual.

[0047] Example 1: Sequence design of novel recombinant collagen

[0048] Human type III collagen alpha 1 chain protein (GenBank accession number: AGL34959.1; collagen type III alpha 1 [Homo sapiens]) was obtained from the NCBI (https: / / www.ncbi.nlm.nih.gov / ) website. According to the human type III collagen sequence, the sequences of sites 297-347, 357-371, 378-407, 462-704, 783-835, 854-884, 897-943, 965-974, 990-1052 and 1137-1172 were selected based on four aspects: retaining key functional domains, maintaining the stability of the triple helix structure of collagen, reducing immunogenicity, improving solubility and facilitating expression, to form a new humanized type III collagen sequence NOP1. The specific amino acid sequence is shown in SEQ ID No. 1, and the corresponding nucleotide sequence encoding NOP1 is shown in SEQ ID Shown in No.3.

[0049] The basis for selecting these specific sequence sites mainly includes the following aspects:

[0050] 1. Retain key functional domains: The selected sites 297-347, 357-371, 378-407, etc., cover the known important functional domains in collagen, such as the region that binds to cell receptors, the key sequence for fiber formation, and the part that interacts with other proteins in the extracellular matrix. Retaining these functional domains can ensure that the recombinant collagen has similar biological functions to natural collagen.

[0051] 2. Maintain the stability of the triple helix structure of collagen: The triple helix structure of collagen is the key to its stability and functionality. Sites 462-704, 783-835 and 1137-1172 were selected, focusing on retaining the amino acid sequence that can promote triple helix formation, especially in the repeating sequence of Gly-XY, which is particularly important.

[0052] 3. Reduce immunogenicity: By selecting specific sequences and excluding areas that may trigger an immune response, the immunogenicity of recombinant collagen in vivo can be reduced. Therefore, shorter sequence segments such as sites 965-974 and 990-1052 were selected to avoid known immunogenic hotspots or areas that react with the immune system.

[0053] 4. Improve solubility and facilitate expression: In order to improve the solubility and facilitate expression of proteins in the Pichia pastoris expression system, specific sequences such as sites 854-884 and sites 897-943 were selected to reduce the aggregation tendency of proteins and ensure that the secretion expression of recombinant collagen is conducive to the expression of Pichia pastoris.

[0054] SEQ ID No.1:

[0055] GPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGSPGSNGAPGQRGEPGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGER GAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENG KPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSPGKDGPPGP AGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPRGSPGPQGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGER

[0056] The humanized type III collagen sequence NOP1 was analyzed using the proteomics database (https: / / web.expasy.org / protparam / ), and it was found that the theoretical pI of the sequence was 9.27, slightly alkaline, with a total of 579 amino acids, a relative molecular weight of 51753.83, an instability index of 28.30, and a grand average of hydropathicity of -1.068.

[0057] Furthermore, on the basis of NOP1, the amino acid sequence and amino acid sites of specific parts of NOP1 were optimized and improved according to indicators such as theoretical isoelectric point, instability coefficient and total hydrophilicity coefficient. Specifically, sites 1-6, GPMGPR were changed to GNEGQP; A at site 8 was changed to Q; R at site 12 was changed to N; R at site 14 was changed to L; L at site 17 was changed to E; GAAGARGNDGARGSD at sites 19-33 was changed to GSNGPMGSRGNPGKN; P at sites 38 and 41 was changed to S; GTAGFP at sites 43-48 was changed to GSPGNA; GSN at sites 55-57 was changed to GNP; A at site 59 was changed to Q; Q at site 62 was changed to E; E at position 65 was changed to K; A at position 68 was changed to N; P at position 72 was changed to A; P at position 74 was changed to E; GIN at position 76-78 was changed to GNP; G at position 83 was changed to N; MGPA at position 87-90 was changed to DGKD; I at position 92 was changed to S; A at position 95 was changed to N; EDGKDGS at position 98-104 was changed to QPGNEGQ; E at position 107 was changed to Q; A at position 110 was changed to Q; L at position 113 was changed to Q; GAAGERGAPGFRGPAGPNGIPGEKGPAGER sequence was removed at positions 115-144; A at position 146 was changed to E; PA at position 149-150 was changed to SN; R at position 153-158 GAAGE was changed to QGSQGN; RDGV was changed to KNGQ at 161-164; G was changed to S at 167; MRGM was changed to SQGS at 170-173; S was changed to N at 176; G was changed to S at 179; ESGRP was changed to KPGNQ at 194-198; P was changed to A at 201; PSGPRGQ was changed to EPGNPGS at 203-209; VMGF was changed to PKGA at 212-215; P was changed to N at 218; NDGA was changed to SPGN at 221-224; NGER was changed to PGNE at 228-231; Q was changed to N at 240; P was changed to Q at 242; 245-249, KNGET was changed to EPGSN; 254-261, PPGPTGPG was changed to SQGNPGKN; 264-269, KGDTGP was changed to PGSPGS; 272-275, PQGL was changed to SPGN; 278, L was changed to S; 282-284, GGP was changed to PGN; 287-290, ENGK was changed to QPGE; 293, E was changed to K; 296-299, PKGD was changed to NQGP; 302, A was changed to E; 305, A was changed to N; 308-314, GKGDAGA was changed to SPGNQGQ; 317-320, ERGP was changed to NKGS;At positions 323-326, LAGA was changed to NPGQ; at positions 329-335, LRGGAGP was changed to NEGQPGQ; at positions 338-341, PEGA was changed to QNGQ; at position 344, L was changed to E; at positions 347-348, IA was changed to SN; at positions 356-360, ERGET was changed to NPGKN; at positions 365-371, PAGFPGA was changed to SPGSQGS; at positions 374-377, QNGE was changed to NQGS; at positions 380-386, GKGERGA was changed to QPGNPGQ; at position 39 0-392, KGE changed to QGK; P changed to N at 395; VK changed to PA at 398-399; RGS changed to PGN at 402-404; G changed to S at 407; AAGF changed to NQGQ at 410-413; ARGL changed to NKGS at 416-419; P changed to N at 422; KD changed to NE at 428-429; VS changed to SN at 446-447; D changed to S at 452; Q changed to N at 455; EK changed to KN at 458-459; At sites 464-467, AQGP was changed to SPGS; at site 470, A was changed to S; at sites 482-485, ERGP was changed to NPGK; at sites 488-491, PQGL was changed to ERGK; at sites 494-497, LAGT was changed to NQGP; at sites 503-506, RDGN was changed to NPGS; at site 509, S was changed to N; at sites 515-516, RD was changed to NK; at sites 521-522, GK was changed to NP; at sites 525-528, RGEN was changed to PGNE; at sites 537-542, PGHPGP was changed to N GQFGE; PR was changed to SN at 545-546; VGPSGP was changed to AGSAGN at 549-554; DGT was changed to NGQ at 558-560; SGH was changed to PGS at 561-563; PIGP was changed to SQGS at 566-569; PRGNR was changed to NKGSP at 572-576; R at 579 was changed to P; GNPGIPGERGKPGNPGAP was added to the tail, and finally another new recombinant collagen sequence NOP2 was designed. The specific amino acid sequence is shown in SEQ ID No. 2, and the corresponding nucleotide sequence encoding NOP2 is shown in SEQ ID No. 4. ;

[0058] SEQ ID No.2:

[0059] GNEGQPGQPGENGLPGEPGSNGPMGSRGNPGKNGQPGSPGSSPGNAGSPGSPGNPGQPGERGKPGNQGPAGEPGNPGSPGNKGEDGKDGSPGNPGQPGNEGQPGQPGQNGQPGEPGSNGPQGSQGNPGKNGQPGSPGSQ GSPGPGSPGSDGKPGPPGSQGKPGNQGPAGEPGNPGSPGPKGAPGNKGSPGNPGKPGNEGGPGGPGPNGQPGEPGSNGPQGSQGNPGKNGDPGSPGSPGSPGNQGSPGTPGNPGQPGEPGKPGNQGPAGEPGNPGSPGNQG QPGNKGSPGNPGQPGNEGQPGQPGQNGQPGEPGSNGPRGSPGNPGKNGPPGSPGSQGSPGNQGSPGQPGNPGQPGEQGKPGNQGPAGEPGNPGSPGNQGQPGNKGSPGNPGSPGNEGPPGPAGNTGAPGSPGSNGPKGSAGN PGKNGSPGSPGSPGSPGGPRGSPGPQGNPGKPGERGKPGNQGPAGEPGNPGSPGNDGLPGNKGSPGNPGDPGNEGSPGAPGANGQFGEPGSNGPAGSAGNPGKNGQPGSPGSQGSPGNKGSPGEPGNPGIPGERGKPGNPGAP

[0060] The NOP2 sequence was analyzed using the proteomics database (https: / / web.expasy.org / protparam / ), and it was found that the theoretical pI of the sequence was 6.70, which was neutral, with a total of 567 amino acids, a relative molecular weight of 51013.81, an instability index of 38.06, and a grand average of hydropathicity of -1.568. In contrast, after optimization, the hydrophilicity and stability of NOP2 increased, and both the instability index and the grand average of hydropathicity were better than those of NOP1. At the same time, the optimization changed the three-dimensional spatial structure and surface properties of the protein. The schematic diagram of the simulated three-dimensional structure of NOP2 is shown below. Figure 1 shown.

[0061] Example 2: Synthesis of DNA sequence, construction of recombinant expression vector, and recombinant Pichia pastoris engineering bacteria

[0062] (1) Synthesis of DNA sequences and construction of recombinant expression vectors: After optimizing the codon preference of Pichia pastoris, the DNA fragments of NOP1 and NOP2 were synthesized by whole gene synthesis (the optimized nucleotide sequences of NOP1 and NOP2 are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively), and Eco RI and Not I recognition sites and signal peptide recognition sites were added to both ends of the gene, respectively. The synthesized gene fragments were cloned into the pPIC9K empty expression vector (purchased from Thermo Fisher Scientific) to obtain recombinant plasmids pPIC9K-NOP1 and pPIC9K-NOP2 expressing NOP1 and NOP2, as shown in FIG. Figure 2 shown.

[0063] (2) Plasmid linearization: Sal I was used to linearize the recombinant plasmid. The enzyme reaction system (100 μL) was as follows: 5 μL Sal I, 10 μL 10*H Buffer, 20 μL recombinant plasmid DNA, 35 μL ddH2O. The reaction conditions were: 37°C for 2-4 hours. At the end of the reaction, the mixture was placed in a 65°C water bath for 15 minutes to inactivate the enzyme. The linearized recombinant plasmid was then recovered using a PCR product purification kit.

[0064] (3) Electroporation and screening of yeast transformants: The linearized plasmid was electroporated into competent cells of Pichia pastoris GS115. Electroporation process: 80 μL of Pichia pastoris GS115 competent cells and 10 μg of linearized recombinant plasmid DNA were transferred to a 0.2 cm ice-precooled electroporation cup; the electroporation cup was placed in an ice bath for 5 min; the sample was electroporated at a voltage of 1500 V, a capacitance of 25 μF, and a resistance of 200 Ω, and the electroporation time was 4 ms; after electroporation, 1 mL of 1 mol / L ice-precooled sorbitol was immediately added to the electroporation cup, mixed, and the contents of the electroporation cup were transferred to a new 1.5 mL EP tube. The bacterial solution was spread on the MD selection plate, and the plate was inverted and cultured at 30°C for 2-3 days until a single colony (positive transformant) appeared. High-copy positive transformants were selected by the G418 gradient method, streaked on the YPD plate, and cultured at 30°C for 2-5 days to obtain two recombinant Pichia pastoris engineered bacteria.

[0065] Example 3: Inducible expression of recombinant Pichia pastoris

[0066] Single colonies of two recombinant Pichia pastoris were picked from the plate and inoculated into 25 mL of BMG. The culture medium was shaken at 30°C and 300 rpm until the OD 600 =2-6; centrifuge at 1500g for 5 min at room temperature, collect cells, remove supernatant, and resuspend cells in BMMY medium to OD 600=1.0, and induce expression; add 20 mL of the above resuspended culture to a 1L shaking flask containing 200 mL of BMMY medium, and continue to grow in a shaking incubator; every 24 hours, add methanol to a final concentration of 0.5% to 1.0% to continue induction; after 96 hours of induction expression, take the culture solution and centrifuge it at 3000g for 5 minutes, take the supernatant, and perform SDS-PAGE electrophoresis. The protein electrophoresis diagram is shown as follows Figure 3 shown.

[0067] Example 4: Purification of recombinant collagen

[0068] The two recombinant Pichia fermentation supernatants of Example 3 were collected, and the supernatants were separated using an ultrafiltration column membrane with a molecular weight cutoff of 10 kDa to remove small molecular weight proteins and salts. The recombinant collagen was then purified by molecular sieve chromatography (filler: Superdex 75prep grade), and pure water was used as the mobile phase at a flow rate of 0.5 mL / min for elution. The samples in the elution peak were collected and freeze-dried to obtain two recombinant collagen lyophilized powders.

[0069] Example 5: Preparation of hydrogel for skin wound healing

[0070] 6 g of recombinant collagen lyophilized powder was dissolved in 100 mL of 20 mM phosphate buffer (pH 5.59) to obtain a recombinant collagen aqueous solution; cellulose nanocrystals (CNC, purchased from Sciencek) were added to pure water at a mass volume ratio of 0.6%, and ultrasonically oscillated for 20 min to obtain a cellulose nanocrystal dispersion; the recombinant collagen aqueous solution was slowly added to the cellulose nanocrystal dispersion at a volume ratio of 1:1 and a flow acceleration of 5 mL / min, and stirred and mixed uniformly at a speed of 50 rpm to obtain a mixed solution; the mixed solution was placed in a hydrochloric acid steam bath and allowed to stand for 12 h to obtain a hydrogel, i.e., a cellulose nanocrystal-recombinant collagen composite hydrogel, such as Figure 4 shown. Figure 4 It showed that recombinant collagen NOP2 and CNC were able to form composite hydrogels with good appearance and mechanical strength, while recombinant collagen NOP1 and CNC failed to form shaped composite hydrogels.

[0071] The mechanical properties of CNC-NOP1 and CNC-NOP2 composite hydrogels were investigated. Figure 5 As shown in the figure, it can be seen that the CNC-NOP2 composite hydrogel has higher and better mechanical properties than the CNC-NOP1 composite hydrogel, which indicates that the CNC-NOP2 composite hydrogel has better mechanical strength.

[0072] Example 6: Mouse wound healing experiment

[0073] A mouse trauma model was established. Each mouse (eight-week-old male C57 mouse, weighing about 23-25 ​​g, purchased from Nanjing Tande Biotechnology Co., Ltd.) was shaved after anesthesia, and a circular skin wound with a diameter of 1 cm was cut on the back with a sterile small blade. Cellulose nanocrystal-recombinant collagen composite hydrogel CNC-NOP2 was applied to the wound, and photos of mouse skin defects were recorded on 0 days, 3 days, 7 days, 10 days, and 14 days (see Figure 2). Figure 6 Wound closure was assessed by estimating wound area (wound healing rate, e.g. Figure 7 shown).

[0074] The experimental results showed that on the third day, the healing rate of the mouse wounds exceeded 60%, and on the 14th day, the healing rate of the mouse wounds reached 99%. This result shows that the cellulose nanocrystal-recombinant collagen composite hydrogel has the effect of quickly healing wounds. In addition, the mouse wounds gradually healed within 14 days, with a significant reduction in wound area and no excessive fibrosis, indicating that the healing process was effective and less scar formation. This shows that the healing process of this experiment has a positive effect on reducing scar formation.

[0075] The present invention provides a recombinant collagen and its application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A recombinant collagen, characterized in that: The recombinant collagen is constructed based on human type III collagen, and its amino acid sequence is shown in SEQ ID No.

2.

2. The nucleic acid molecule encoding the recombinant collagen according to claim 1, characterized in that: Its nucleotide sequence is shown as SEQ ID No.

6.

3. A recombinant expression vector, characterized in that: Contains the nucleotide sequence of the nucleic acid molecule according to claim 2.

4. A recombinant strain, characterized in that: The host is Pichia pastoris, and the recombinant expression vector according to claim 3 is contained.

5. Use of the recombinant collagen according to claim 1 in preparing a product for healing skin wounds.

6. The use according to claim 5, characterized in that: The skin wound healing product is a hydrogel.

7. The use according to claim 6, characterized in that: The hydrogel is obtained by adding a recombinant collagen solution into a cellulose nanocrystal dispersion, mixing the mixture, and placing the mixture in a hydrochloric acid steam bath for standing.

8. The use according to claim 7, characterized in that: The concentration of the recombinant collagen solution is 40-80 g / L, and the solvent is 10-30 mM phosphate buffer with a pH of 4.49-6.

80.

9. The use according to claim 7, characterized in that: The cellulose nanocrystal dispersion is prepared by using cellulose nanocrystals as a supporting framework, adding cellulose nanocrystals into pure water at a mass volume ratio of 0.2-1.0%, and then subjecting the mixture to ultrasonic oscillation.

10. The use according to claim 7, characterized in that: The volume ratio of the recombinant collagen solution to the cellulose nanocrystal dispersion is 2:1-1:3; the flow rate of the adding is 2-10 mL / min; and the standing time is 6-18 hours.

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