Recombinant collagen type iii, its preparation method and application

Abstract

This invention discloses recombinant type III collagen, its preparation method, and its applications. The amino acid sequence of the recombinant type III collagen is shown in SEQ ID NO: 9. The recombinant type III collagen integrates the RGDV highly active motif and the EKEK hydrophilic stable domain with the core structure of type III collagen to achieve an integrated design of "high activity and long-lasting effect". The in vitro cell migration and collagen secretion effects are improved by more than 50% compared with wild type. Through anti-enzyme cleavage site modification and hydrophilic modification, the in vivo residence half-life is significantly prolonged to meet the long-term repair needs of the dermis.

Description

Technical Field

[0001] This invention relates to the field of recombinant protein technology, specifically to recombinant type III collagen, its preparation method, and its applications. Background Technology

[0002] Collagen is a core component of the human extracellular matrix. Type III collagen (COL3A1) is mainly distributed in the dermis, blood vessel walls, and embryonic tissues, playing an irreplaceable role in skin rejuvenation, wound healing, and dermal matrix regeneration. Currently, the sources and applications of collagen-based biomaterials face the following key technological bottlenecks: 1. Inherent defects of animal-derived collagen: Traditional medical collagen is mostly extracted from animal tissues such as bovine Achilles tendon and pig skin, which presents three major problems: ① Immunogenicity risk: Animal-derived collagen sequences differ from human collagen, which can easily trigger an immune rejection response; ② Pathogen contamination risk: Animal tissues may carry pathogens such as viruses and prions, and the risk of cross-infection cannot be completely ruled out; ③ Limited function: Naturally extracted collagen lacks targeted activity modification, resulting in limited repair efficiency. 2. Insufficient Performance of Existing Recombinant Collagen: To address the shortcomings of animal-derived materials, recombinant collagen has become a research hotspot, but existing products still have limitations: ① Poor in vivo stability: Conventional recombinant type III collagen is easily degraded rapidly by matrix metalloproteinases (such as MMP-1) in vivo, with a residence time at the site of action of only 3-7 days, which is insufficient to meet the long-term regeneration needs of the dermis; ② Limited bioactivity: It has not been targeted to integrin receptors, resulting in weak promotion of fibroblast adhesion, proliferation, and collagen synthesis; ③ Imperfect expression technology: Prokaryotic systems cannot achieve the essential hydroxyproline modification for collagen, leading to protein folding errors; mammalian cell (such as CHO) culture is costly and difficult to scale up; although insect cell systems can perform hydroxylation modification, they lack fine-grained control over protein folding efficiency and modification adequacy, resulting in low yields of proteins with correct triple helix structures; 3. Special needs for dermal skin repair: New medical aesthetic and wound repair technologies such as microneedling and mesotherapy require materials to remain in the dermis for a long time and continue to exert biological activity. However, the "short half-life" and "low activity" characteristics of existing collagen materials cannot meet the application requirements of this scenario.

[0003] Therefore, developing a "highly active, long-lasting, stable, and process-controllable" human type III recombinant collagen is of great significance for promoting technological upgrades in the fields of skin regenerative medicine and aesthetic medicine. Summary of the Invention

[0004] This invention provides a human type III recombinant collagen protein that meets the requirements of high activity and long-term stability, satisfying the application needs of skin regeneration medicine and medical aesthetics.

[0005] In view of this, the solution of the present invention is as follows: A first aspect of the present invention is to provide recombinant type III collagen having the amino acid sequence shown in SEQ ID NO: 9.

[0006] A second aspect of the invention is to provide a nucleic acid molecule encoding the recombinant type III collagen described in the first aspect.

[0007] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 8.

[0008] A third aspect of the present invention is to provide a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0009] Furthermore, the recombinant expression vector was constructed based on the pFastBac1 vector.

[0010] A fourth aspect of the present invention is to provide an engineered cell that expresses the recombinant type III collagen of the first aspect, or the nucleic acid molecule of the second aspect, or the recombinant expression vector of the third aspect.

[0011] Furthermore, the engineered cells are obtained by first constructing a recombinant baculovirus based on a recombinant expression vector, and then infecting insect cells.

[0012] The fifth aspect of the present invention is to provide a method for preparing recombinant type III collagen, wherein a recombinant expression vector is constructed based on the nucleotide sequence shown in SEQ ID NO: 8, and then packaged into a recombinant baculovirus and infected with insect cells for culture, the culture supernatant is collected, and the recombinant type III collagen is separated and purified.

[0013] Furthermore, the recombinant baculovirus is obtained by recombining the recombinant expression vector in DH10Bac competent cells and then packaging it in Sf9 insect cells.

[0014] Furthermore, the insect cells are High Five insect cells.

[0015] Furthermore, the culture process was carried out in SF-900 III medium supplemented with L-ascorbic acid and FeSO4.

[0016] A sixth aspect of the invention is to provide a skin regeneration and repair product comprising the recombinant type III collagen described in the first aspect.

[0017] Furthermore, the skin regeneration and repair products include medical devices, medical dressings, injectable products, etc., but are not limited to microneedling, mesotherapy, wound repair, skin regeneration scaffold products, or cosmetics.

[0018] A seventh aspect of the present invention is to provide the application of the recombinant type III collagen described in the first aspect in the preparation of skin regeneration and repair materials.

[0019] Furthermore, the skin regeneration and repair materials include, but are not limited to, microneedles, mesotherapy, wound repair dressings, skin regeneration scaffold materials, or cosmetic ingredients.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The recombinant type III collagen provided by this invention integrates the highly active RGDV motif, the EKEK hydrophilic stable domain, and the core structure of type III collagen to achieve an integrated design of "high activity and long-lasting effect". The in vitro cell migration and collagen secretion effects are improved by more than 50% compared with wild type. Through anti-enzyme cleavage site modification and hydrophilic modification, the in vivo residence half-life is significantly extended to meet the long-term repair needs of the dermis.

[0021] The recombinant type III collagen provided by this invention is designed based on human sequence, contains no animal-derived components, and has extremely low immunogenicity (hemolysis rate ≤1%, allergic reaction rate ≤1%). It is suitable for various dermal layer application scenarios such as microneedling, mesotherapy, and wound repair, taking into account both medical aesthetic and medical needs, and has high market application value.

[0022] The method for preparing recombinant type III collagen provided by this invention increases the protein yield with the correct triple helix structure by more than 30% compared with conventional insect cell expression methods, and the hydroxyproline content reaches 13.8%, which is close to the natural level. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the functional domain structure design of the recombinant collagen HL-RHC-III described in this invention.

[0024] Figure 2 The image shows the recombinant baculovirus transfer vector pFB-HL-RHC-III constructed in this invention.

[0025] Figure 3 The results of SDS-PAGE and Western Blot analysis of the HL-RHC-III expression and purification process in Example 5 of this invention are shown.

[0026] Figure 4 The images show the SEC-HPLC chromatogram and circular dichroism chromatogram of HL-RHC-III in Example 5 of this invention.

[0027] Figure 5 A bar chart comparing the fluorescence quantitative results of the cell adhesion experiment in Example 6 of this invention ( p < 0.001).

[0028] Figure 6 This is a graph showing the change in the residual rate of HL-RHC-III and control proteins over time in the anti-collagenase degradation experiment of Example 6 of the present invention.

[0029] Figure 7 This is a quantitative curve of the fluorescence signal intensity of two groups at different time points after subcutaneous implantation in rats in Example 6 of the present invention.

[0030] Figure 8 Results of HE staining and Masson staining in Example 7 of the invention. Detailed Implementation

[0031] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] The recombinant collagen designed in this invention (named HL-RHC-III) contains the following functional domains sequentially from the N-terminus to the C-terminus (see structural diagram). Figure 1 Each domain is connected via flexible linker peptides or directly tandem to ensure spatial conformation compatibility. 1. Signal peptide (SEQ ID NO:1): Bee venom peptide signal peptide MKFLVNVALVFMVVYISYIYA, which is responsible for guiding the recombinant protein to be secreted into the extracellular space, simplifying downstream purification; 2. Purification tag (SEQ ID NO: 2): 6×His tag HHHHHH (coding sequence caccaccaccaccaccac), for Ni 2+ -NTA affinity chromatography purification; 3. Flexible linker peptide (SEQ ID NO: 3): (GGGGS)3 sequence GGGGSGGGGSGGGGS, avoids steric hindrance from adjacent functional domains and ensures correct protein folding; 4. Activity-enhancing domain (SEQ ID NO: 4): RGDV integrin-binding motif RGDV, this variant has a high affinity for integrin αvβ3 and α5β1, significantly promoting fibroblast adhesion, spreading and collagen secretion; 5. Main collagen domain (SEQ ID NO: 5): contains 10 (GXX) repeating units, with the sequence GPP-GPP-GPM-GPP-GPP-GPM-GPP-GPP-GPM-GPP, of which 3 GPM units (proline→hydroxyproline modification sites) enhance the stability of the triple helix structure, and the remaining GPP units ensure structural rigidity; 6. Long-lasting stable domain (SEQ ID NO: 6): The hydrophilic oligopeptide chain EKEKEKEK is negatively charged and highly hydrophilic, increasing the protein hydration radius and slowing down in vivo clearance; at the same time, some "-GY-" units in the main structure are replaced with "-GP-", reducing the recognition efficiency of the MMP-1 restriction site; 7. Trimerization and secretion domain (SEQ ID NO: 7): Native C-terminal peptide sequence of human type III collagen (amino acids 1212-1466 of UniProt P02461) CCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLDAIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL is responsible for guiding the assembly and secretion of collagen trimers.

[0033] Full-length amino acid sequence of HL-RHC-III (functional domains in tandem, SEQ ID NO: 9): MKFLVNVALVFMVVYISYIYAHHHHHHGGGGSGGGGSGGGGSRGDVGPPGPPGPMGPPGPPGPMGPPGPPGPMGPPEKEKEKCCGGVGAAAIAGIGGEKAGGFAPYYGDEPMDFKINTDEIMTSLKSVNGQIESLISPDGSRKNPARNCRDLKFCHPELKSGEYWVDPNQGCKLD AIKVFCNMETGETCISANPLNVPRKHWWTDSSAEKKHVWFGESMDGGFQFSYGNPELPEDVLDVHLAFLRLLSSRASQNITYHCKNSIAYMDQASGNVKKALKLMGSNEGEFKAEGNSKFTYTVLEDGCTKHTGEWSKTVFEYRTRKAVRLPIVDIAPYDIGGPDQEFGVDVGPVCFL Optimized coding gene sequence (SEQ ID NO: 8): targeting the fall armyworm ( Spodoptera frugiperda Insect cell codon preference optimization, including the reverse translation sequence of the above full-length amino acid sequence, with BamHI and EcoRI restriction sites introduced at both ends, the sequence is as follows: The recombinant collagen HL-RHC-III provided by this invention is the first to integrate the highly active RGDV motif, the EKEK hydrophilic stable domain, and the core structure of type III collagen, achieving an integrated design of "high activity and long-lasting effect." Its in vitro cell migration and collagen secretion promotion effects are more than 50% higher than those of wild-type recombinant collagen. Through anti-enzyme cleavage site modification and hydrophilic modification, its in vivo residence half-life reaches 18.5 days, which is 2.8 times longer than that of conventional recombinant collagen (7.2 days), meeting the long-term repair needs of the dermis.

[0034] The recombinant collagen HL-RHC-III provided by this invention is designed based on human sequence, contains no animal-derived components, and has extremely low immunogenicity (hemolysis rate ≤1%, allergic reaction rate ≤1%).

[0035] This invention also provides a method for preparing the above-mentioned recombinant collagen HL-RHC-III, comprising: 1. Construction of recombinant expression vectors and preparation of recombinant baculoviruses 1) Vector construction: The gene encoding SEQ ID NO: 8 was cloned downstream of the polyhedral protein promoter (pPH) of the baculovirus transfer vector pFastBac1. The recombinant transfer plasmid pFB-HL-RHC-III was constructed by inserting the gene into the BamHI and EcoRI double restriction sites (see Figure 2 for vector map). The correctness of the cloning was verified by DNA sequencing. 2) Preparation of recombinant bacmids: pFB-HL-RHC-III was transformed into DH10Bac competent cells. Positive colonies containing recombinant bacmids Bacmid-HL-RHC-III were obtained by blue-white screening (X-Gal / IPTG) and antibiotic resistance screening (kanamycin, tetracycline, gentamicin). Bacmid DNA was extracted. 3) Recombinant baculovirus packaging and amplification: Sf9 insect cells cultured on adherent walls were transfected with Bacmid-HL-RHC-III using Cellfectin II liposome transfection reagent (cultured at 28℃ for 4-5 days). The supernatant was collected to obtain P1 generation recombinant baculovirus (vHL-RHC-III). The virus titer was determined by plaque assay, and the virus was amplified to P3 generation in Sf9 suspension cells to obtain high-titer working solution (titer > 1×10⁻⁶). 8 pfu / mL).

[0036] 2. Optimized insect cell expression and fermentation process

[0037] 1) Cell culture: High Five insect cells were cultured in suspension in SF-900 III serum-free medium at 28℃ and 110 rpm until mid-logarithmic growth phase (cell density 2.0 × 10⁶ cells / year). 6 (cells / mL, viability > 95%) 2) Viral infection: Inoculate with P3 generation vHL-RHC-III virus at a multiplicity of infection (MOI) of 3; 3) Optimization of expression technology: ① Addition of cofactors: Immediately after infection, L-ascorbic acid (final concentration 50 µg / mL) and FeSO4 (final concentration 10 µM) were added to the culture medium to promote prolyl-4-hydroxylase activity and improve hydroxyproline modification efficiency. ②Temperature control: 12 hours after infection, the culture temperature was lowered from 28℃ to 25℃ to slow down cell metabolism and reduce protein misfolding.

[0038] 4) Protein harvesting: 72 hours after infection, the supernatant was collected by centrifugation at 4000g for 20 minutes at 4℃. The supernatant contained secreted HL-RHC-III protein.

[0039] 3. Purification and refolding of recombinant collagen

[0040] 1) Supernatant pretreatment: Filter the supernatant through a 0.45 µm filter membrane, add a mixture of protease inhibitors (Roche) and 1 mM PMSF; concentrate 20-fold through a tangential flow ultrafiltration system with a molecular weight cutoff of 10 kDa, and replace the buffer with Binding Buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0). 2) Metal chelate affinity chromatography: The sample was loaded onto a Ni-NTA agarose gel column (5 mL column bed volume), equilibrated with Binding Buffer, and then washed sequentially with 10 column volumes of Binding Buffer and Wash Buffer (20 mM Tris-HCl, 300 mM NaCl, 50 mM imidazole, pH 8.0). Finally, it was eluted with Elution Buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 8.0), and the 280 nm UV absorption peak was collected. 3) Size exclusion chromatography: Load affinity chromatography eluent onto a HiLoad 16 / 600 Superdex 200 pg gel filter column (equilibrated with PBS pH 7.4), elute at a flow rate of 1 mL / min, and collect the target protein peak in the form of trimer (excluding aggregates and monomers). 4) Concentration and storage: Concentrate to 5-10 mg / mL using 30 kDa ultrafiltration centrifuge tubes, sterilize by filtration through a 0.22 µm filter membrane, and aliquot and store at -80℃.

[0041] The above preparation method, through co-expression of prolyl-4-hydroxylase subunit, optimization of culture temperature and addition of cofactors, resulted in a protein yield with the correct triple helix structure that was more than 30% higher than that of conventional insect cell expression methods, with a hydroxyproline content of 13.8% (close to the natural level).

[0042] This invention also provides applications of the recombinant collagen HL-RHC-III described above, which is suitable for various dermal layer treatment scenarios such as microneedling, water-light injection, and wound repair, taking into account both medical aesthetic and medical needs, and has high market application value.

[0043] In a preferred embodiment, the application includes, but is not limited to, the following: 1) Injectable filler gel: HL-RHC-III protein is mixed with genipin crosslinking agent to prepare injectable hydrogel for facial wrinkle filling and skin laxity repair (water injection / microneedling). 2) Wound repair dressings: Recombinant collagen is combined with hyaluronic acid and chitosan to prepare porous sponge or hydrogel dressings for dermal regeneration and repair of chronic wounds and burn wounds. 3) Skin regeneration scaffold: Tissue engineering scaffolds are prepared by combining PLGA with freeze-drying technology for dermal replacement of skin defects.

[0044] Example 1: Construction of recombinant transfer plasmid pFB-HL-RHC-III

[0045] 1. Gene synthesis: The gene encoding SEQ ID NO: 8 was synthesized by Nanjing Genscript Biotech Co., Ltd., with BamHI and EcoRI restriction sites at both ends; 2. Double enzyme digestion reaction: Take the synthesized gene fragment (10 μg) and pFastBac1 vector (5 μg), add BamHI (10 U) and EcoRI (10 U) respectively, and digest at 37℃ for 2 hours; 3. Fragment recovery: The enzyme digestion products were separated by 1% agarose gel electrophoresis, and the target gene fragment (approximately 2.2 kb) and vector fragment (approximately 4.8 kb) were recovered using the Qiagen DNA Gel Recovery Kit. 4. Ligation and transformation: Mix the target gene and vector at a molar ratio of 3:1, add T4 DNA ligase (5 U), and ligate overnight at 16°C; transform the ligation product into DH5α competent cells, plate on LB agar plates containing ampicillin, and incubate at 37°C for 12 hours; 5. Verification: Single colonies were picked and plasmids were extracted. The plasmids were verified by double enzyme digestion (releasing the 2.2 kb target fragment) and DNA sequencing, confirming that the recombinant plasmid pFB-HL-RHC-III was constructed correctly.

[0046] Example 2 Packaging and amplification of recombinant baculovirus

[0047] 1. Transformation of pFB-HL-RHC-III plasmid (2 μg): Transform DH10Bac competent cells, plate them on LB plates containing X-Gal, IPTG, kanamycin, tetracycline and gentamicin, and incubate at 37°C for 48 hours. 2. Extraction of recombinant Bacmid-HL-RHC-III: White positive colonies were picked and inoculated into LB liquid medium containing three antibiotics. The medium was incubated at 37°C with shaking for 12 hours. Bacmid-HL-RHC-III was extracted using the Qiagen Bacmid Extraction Kit. 3. Virus packaging: Take Sf9 cells (1×10⁻⁶) 6 Cells (cells / well) were seeded into 6-well plates and cultured adherently for 2 hours; 2 μg Bacmid-HL-RHC-III was mixed with 5 μL Cellfectin II and incubated at room temperature for 15 minutes, then added to the cell wells and cultured at 28°C for 4-5 days. The supernatant was collected to obtain the P1 generation virus. 4. Viral amplification: P1 generation virus was used to infect 10 mL of Sf9 suspension cells (1×10⁻⁶ cells) with an MOI of 0.1. 6 The virus was cultured at 28°C and 110 rpm for 72 hours to obtain P2 generation virus. Then, 100 mL of Sf9 cells were infected with MOI=1 to amplify and obtain P3 generation virus. The titer, as detected by plaque assay, was 1.5 × 10⁻⁶ cells / mL. 8 pfu / mL.

[0048] Example 3: Insect cell expression and fermentation of HL-RHC-III

[0049] 1. Cell culture: Add 200 mL of SF-900 III medium to a 500 mL shake flask and seed High Five cells to an initial density of 5 × 10⁶ cells / mL. 5 Cells / mL, cultured at 28℃ and 110 rpm until 2.0 × 10⁻⁶ cells / mL.6 cells / mL (viability 96%) 2. Viral infection: Add 600 μL of P3 generation virus solution (MOI=3) and gently shake to mix; 3. Process optimization: L-ascorbic acid (final concentration 50 µg / mL) and FeSO4 (final concentration 10 µM) were added immediately after infection; the culture temperature was adjusted to 25℃ 12 hours after infection. 4. Protein harvesting: 72 hours after infection, centrifuge at 4℃ and 4000g for 20 minutes, collect the supernatant (about 180 mL), and detect the expression of the target protein (about 75 kDa monomer) by SDS-PAGE.

[0050] Example 4: Purification and refolding of HL-RHC-III

[0051] 1. Supernatant pretreatment: Filter the supernatant through a 0.45 µm filter membrane, add a mixture of protease inhibitors and 1 mM PMSF; concentrate to 9 mL by tangential flow ultrafiltration, and replace with Binding Buffer 3 times; 2. Affinity chromatography: After equilibrating the Ni-NTA column with Binding Buffer, load the sample at a flow rate of 1 mL / min; wash with Binding Buffer until the baseline is stable, wash with Wash Buffer to elute contaminating proteins, and elute the target protein with Elution Buffer, collecting the 280 nm absorption peak (elution volume approximately 8 mL). 3. Gel filtration chromatography: The HiLoad 16 / 600 Superdex 200 pg column was equilibrated with PBS, 5 mL of affinity elution buffer was loaded, and elution was performed at a flow rate of 1 mL / min. The main peak (trimeric form) in the 150-669 kDa range was collected. 4. Concentration and sterilization: Concentrate to 8 mg / mL by ultrafiltration, filter through a 0.22 µm filter membrane, and aliquot and store at -80℃; the protein concentration was determined by BCA method to be 7.8 mg / mL, with a yield of approximately 65%.

[0052] Example 5: Identification and Quality Analysis of HL-RHC-III

[0053] 1. SDS-PAGE and Western Blot: Under reducing conditions, a single main band (approximately 75 kDa) is observed; under non-reducing conditions, the main band > 180 kDa (trimer); Western Blot can specifically recognize both anti-His-tagged antibodies and anti-human type III collagen antibodies (see...). Figure 3 ); 2. SEC-HPLC analysis: Detected using a TSKgel G4000SWxl column, a single symmetrical peak was observed. The retention time corresponds to the trimer molecular weight (see...). Figure 4 ); 3. Circular dichroism analysis: Negative ellipticity at 197 nm and positive ellipticity at 221 nm, consistent with the triple helix structure of collagen; melting temperature (Tm) is 42.5℃ (see...). Figure 4 ); 4. Amino acid analysis: Hydroxyproline content was 13.8%, consistent with the modification level of natural collagen; 5. Mass spectrometry verification: The molecular weight measured by MALDI-TOF mass spectrometry is consistent with the theoretical value, and LC-MS / MS peptide mapping verifies the presence of RGDV, GPM and C-terminal peptide characteristic sequences.

[0054] Example 6: Verification of Biological Functions

[0055] (1) Cell adhesion activity experiment

[0056] Methods: HL-RHC-III (experimental group) and RGDV-free recombinant collagen (control group) were coated at 10 µg / mL into 96-well plates and incubated overnight at 4°C; human dermal fibroblasts (HDFs, 1×10⁶ cells / well) were seeded. 4 Cells / well), incubated at 37℃ for 2 hours; Calcein-AM staining, and fluorescence intensity of live cells was quantified using a fluorescence microplate reader; Results: The fluorescence intensity in the experimental group was 1.9 times that of the control group (p<0.001, see...). Figure 5 This demonstrates that the RGDV domain significantly enhances cell adhesion.

[0057] (2) Anti-collagenase degradation experiment

[0058] Methods: Equal amounts of HL-RHC-III and control protein were incubated with 0.1 µg / mL MMP-1 at 37℃. Samples were taken at 0, 2, 4, 8, and 24 hours. The residual rate of full-length protein was detected by SDS-PAGE and quantified by ImageJ. Results: After 24 hours, the HL-RHC-III residue rate was 65%, compared to 20% in the control group. The degradation half-life was 2.8 times that of the control group (see...). Figure 6 The ability to resist enzymatic degradation is significantly improved.

[0059] (3) Long-term effect experiment of subcutaneous implantation in rats

[0060] Methods: Hydrogels containing HL-RHC-III and control protein were cross-linked with genipin to prepare hydrogels, which were then labeled with DIR fluorescence and implanted subcutaneously into the back of SD rats (200 µL / rat); the fluorescence signal intensity was detected by in vivo imaging at regular postoperatively. Results: The in vivo residence half-life of HL-RHC-III was 18.5 days in the group and 7.2 days in the control group (see...). Figure 7 ), and its stability in vivo is significantly prolonged.

[0061] Example 7 Application Example - Preparation and Effects of Injectable Filler Gel

[0062] 1. Gel preparation: Mix HL-RHC-III protein solution (8 mg / mL) with genipin (final concentration 0.5 mM) and incubate at 37°C for 30 minutes to form an injectable hydrogel (viscosity 500-800 mPa). s); 2. Animal experiments: SD rats were injected with gel (100 µL / rat) in the back. Four weeks post-surgery, skin tissue from the injection site was collected for HE staining and Masson staining. Results are as follows: Figure 8 As shown.

[0063] 3. Results: The dermal layer thickness at the injection site increased by 1.8 times compared to the control group ( Figure 8 (See the image below). The collagen fibers are arranged in a regular manner with no obvious inflammatory reaction, proving that it has excellent skin regeneration and repair effects.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Recombinant type III collagen, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

9.

2. A nucleic acid molecule encoding the recombinant type III collagen of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

8.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector was constructed based on the pFastBac1 vector.

6. An engineered cell, characterized in that, The engineered cells express the recombinant type III collagen of claim 1, or the nucleic acid molecule of claim 2 or 3, or the recombinant expression vector of claim 4 or 5.

7. A method for preparing recombinant type III collagen, characterized in that, A recombinant expression vector was constructed based on the nucleotide sequence shown in SEQ ID NO: 8, then packaged into a recombinant baculovirus and infected with insect cells for culture. The culture supernatant was collected and purified to obtain the recombinant type III collagen.

8. The preparation method according to claim 7, characterized in that, The recombinant baculovirus was obtained by recombination of a recombinant expression vector in DH10Bac competent cells and packaging in Sf9 insect cells; and / or, the infected insect cells were High Five insect cells.

9. A skin regeneration and repair product, characterized in that, It contains the recombinant type III collagen as described in claim 1.

10. The use of the recombinant type III collagen according to claim 1 in the preparation of skin regeneration and repair materials.

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