Repetitive sequence recombinant III type collagen and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to repetitive sequence recombinant III type collagen and application thereof. According to the repetitive sequence recombinant III type collagen, a 438-512 peptide fragment, a 565-639 peptide fragment and a 940-1002 peptide fragment in a human III type collagen are respectively subjected to five repetitions, and the C tail end is connected with a 1189-1200 fragment of the III type collagen, so that the repetitive sequence recombinant III type collagen CAIII-1, CAIII-2 and CAIII-3 is obtained. The repetitive sequence recombinant III type collagen has great advantages in the aspects of cell migration, safety and the like, and can be used as a raw material for preparing wound dressings and skin care products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a repetitive sequence recombinant type III collagen and an application thereof. Background Art

[0002] Collagen is the most abundant protein in mammalian tissues, accounting for 30% of the total protein content of the body. It is also an important component of the extracellular matrix, maintaining the structure and function of tissues and organs such as skin, cartilage, tendons, ligaments and internal organs. It has the characteristics of biodegradability, low immunogenicity and promoting cell proliferation and differentiation, and is an ideal biomedical and beauty skin care material. Collagen is a fibrous protein assembled from multiple tropocollagens. Tropocollagen is the basic unit of collagen and consists of 3 polypeptide chains. The primary structure of the tropocollagen peptide chain has (Gly-XY)n repeating units, in which X and Y are often proline and hydroxyproline, and a few are lysine and hydroxylysine. Proline and hydroxyproline can cause the triple helix structure of collagen to twist sharply, thereby improving the stability of collagen.

[0003] At present, the collagen genes identified in vertebrates encode about 29 types of collagen. Based on the primary structure, the length of the triple helical domain, the molecular weight, the interruption of the triple helical structure and the size and shape of the terminal domain, collagen can be divided into four major categories: the first type is fibrillar collagen, which includes types I, II, III, V, XI, XXIV and XXVII, among which types I, II and III are more widely distributed in vertebrates, while types V and XI are less distributed, but they play an auxiliary role in the assembly of types I, II and III; the second type is triple helical fibrillar collagen (FACIT), which includes types IX, XII, XIV, XVI, XIX and XXII. FACIT collagen does not form collagen, but can interact with fibrillar collagen, regulate the formation and size of collagen fibers, and control collagen synthesis in the extracellular matrix; the third type of reticular collagen, there are types IV, VII and XXVIII. Among them, type IV collagen forms a fibrous reticular structure, while type VII collagen assembles into anchoring fibers that connect the epidermis to the dermis; the fourth type of interrupted helical collagen (MACITs), there are types XⅢ, XXⅢ and XXV. Among them, XⅢ, XXⅢ and XXV collagens are type II transmembrane proteins, which are composed of a hydrophobic transmembrane domain, a short N-terminal cytoplasmic domain and an extracellular domain composed of three types of collagen. Among the above-mentioned collagens, human type III collagen is encoded by the COL3A1 gene, and the Gly-XY sequence is highly repetitive. It is mainly distributed in the reticular structure of the skin, blood vessel walls and most tissues, making the organs elastic, resistant to tension and maintaining the integrity of the organs. The adult dermis no longer has the ability to synthesize type III collagen. External supplementation of type III collagen can achieve the effects of repairing wrinkles, moisturizing, and tightening the skin. Therefore, type III collagen is highly respected in the field of beauty and skin care. In addition, type III collagen also plays an important role in cell binding, hemostasis and angiogenesis, tissue remodeling, fetal development and wound healing.

[0004] The acquisition of collagen is generally divided into extraction and preparation methods. Collagen extracted from animal sources still has many disadvantages such as immunogenicity, poor uniformity, and hidden dangers of infectious diseases. With the rapid development of molecular biology technologies such as genetic engineering and protein engineering, the research on recombinant collagen produced and prepared by emerging technologies is also rapidly advancing. Compared with traditional extraction methods, recombinant collagen products have great advantages in purity, stability, activity and safety. They can not only avoid the risks of traditional extraction methods, but also improve other properties of collagen (such as hydrophilicity). In addition, due to sequence reasons, it is difficult to express full-length natural collagen. Therefore, the collagen obtained by repeating or combining fragments of collagen with different functions has the advantages of easy expression and stronger target functionality, thus gaining more attention and application. Summary of the invention

[0005] Based on the above problems, the present invention provides a repetitive sequence recombinant type III collagen. The repetitive sequence recombinant type III collagen of the present invention is obtained by repeating 5 peptide segments 438-512, 565-639, and 940-1002 in human type III collagen, respectively, and connecting the type III collagen 1189-1200 fragment to the C-terminus. The obtained repetitive sequence recombinant type III collagen CAⅢ-1, CAⅢ-2, and CAⅢ-3 show great advantages in cell migration, safety, etc., and can be used as raw materials for the preparation of wound dressings and skin care products.

[0006] The repetitive sequence recombinant type III collagen of the present invention comprises CAⅢ-1, CAⅢ-2 and CAⅢ-3.

[0007] Among them, the repetitive sequence recombinant type III collagen CAⅢ-1 is composed of 387 amino acids, and the basic repeating unit is GAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAP, which is a peptide segment from 438 to 512 of human type III collagen, with 5 repeats, and the C-terminus contains the type III collagen 1189-1200 fragment, and the amino acid sequence is as follows: GAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGA AGERGAPGFRGPAGPNGIPGEKGPAGERGAPGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPPPGAPGPCCGGV; Among them, the repetitive sequence recombinant type III collagen CAⅢ-2 is composed of 387 amino acids, and the basic repeating unit is PPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQG, which is a peptide segment from human III collagen 565-639, 5 repeats, and the C-terminus contains the type III collagen 1189-1200 fragment, and the amino acid sequence is as follows: PPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPP GKNGETGPQGPPGPTGPGGDKGDTGPPGPQGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGPPGAPGPCCGGV; Among them, the repetitive sequence recombinant type III collagen CAⅢ-3 is composed of 327 amino acids, and the basic repeating unit is PPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPG, which is derived from the peptide segment 940~1002 of human III collagen, 5 repeats, and the C-terminus contains the type III collagen 1189-1200 fragment, and the amino acid sequence is as follows: PPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVK GESGKPGANGLSGERGPPGPQGLPGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGPPGAPGPCCGGV.

[0008] Preferably, the DNA sequences of the repetitive sequence recombinant type III collagen CAⅢ-1, CAⅢ-2, and CAⅢ-3 correspond to SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0009] Preferably, in the prokaryotic expression method of recombinant type III collagen of the repetitive sequence, the expression vector is pET28a(+), and the expression is carried out by the Escherichia coli expression strain, and the expression strains include: BL21(DE3), BL21(DE3)pLysS, BL21Star(DE3), BL21Star(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, OrigamiB(DE3), OrigamiB(DE3)pLysS, SHuffle T7, BLR(DE3), BLR(DE3)pLysS, B834(DE3), B834(DE3)pLysS.

[0010] Preferably, in the eukaryotic expression method of recombinant type III collagen of the repetitive sequence, the expression vector is pcDNA3.1(+) or pFastBac1, and the eukaryotic cells are used for expression, and the expression cells include: Expi293, ExpiCHO, and Expi sf9.

[0011] Preferably, the expression vector of the repetitive sequence recombinant type III collagen contains a histidine tag and a fusion tag. The histidine tag preferably contains 6-8 histidines, and the fusion tag preferably contains MBP or GFP.

[0012] The application of the repetitive sequence recombinant type III collagen described in the present application is to use the repetitive sequence recombinant type III collagen CAⅢ-1, CAⅢ-2, and CAⅢ-3 as raw materials for the preparation of wound dressings and skin care products.

[0013] The beneficial effects of the present invention are as follows: The repetitive sequence recombinant type III collagen CAⅢ-1 of the present invention contains a DGKDG peptide segment, which has been verified to be related to caspase 3-7. Caspase plays a key role in the process of cell apoptosis and can promote cell apoptosis and skin repair when used in skin care products. The repetitive sequence recombinant type III collagen CAⅢ-2 contains a RGQPGVMGF peptide segment, which is one of the main F residue cross-fiber clusters in type III collagen and is a key component of the tyrosine kinase receptor DDR2, the cysteine-rich secretory acidic protein SPARC and the vWF-collagen binding complex. The repetitive sequence recombinant type III collagen CAⅢ-2 also contains a KNGETGPQGP peptide segment, which has been verified to be related to matrix metalloproteinase 3 (MMP-3). Matrix metalloproteinases have been shown to cleave collagen III along the molecular direction and activate other matrix lysins, which are known to play an important role in physiological processes such as tissue remodeling, wound healing, and inflammation elimination. The repetitive sequence recombinant type III collagen CAⅢ-3 contains a GPLGIAGITGARGLA peptide segment, which has been verified to be related to matrix metalloproteinases 1, 3, and 9. In addition, the frequency of GXY in the repetitive sequence recombinant type III collagen CAⅢ-1, CAⅢ-2, and CAⅢ-3 of the present invention is low, and the hydroxylation of proline mainly occurs in them, which is conducive to the correct expression of the corresponding sequence. On this basis, the present invention screened the 438-512 peptide segment, the 565-639 peptide segment, and the 940-1002 peptide segment in human type III collagen, and finally selected the above peptide segments after multiple experiments. The peptide segments were repeated 5 times, and the C-terminus was connected to the type III collagen 1189-1200 fragment. The repetitive sequence recombinant type III collagen CAⅢ-1, CAⅢ-2, and CAⅢ-3 finally obtained showed great advantages in cell migration and safety, and can be used as raw materials for the preparation of wound dressings and skin care products. In addition, the repetitive sequence recombinant type III collagen CAⅢ-1, CAⅢ-2, and CAⅢ-3 of the present invention can be prepared not only by prokaryotic expression methods, but also by eukaryotic expression methods. Combined with the application of histidine tags and fusion tags, large-scale expression can be achieved. In addition, CAⅢ-1-GFP prepared by the eukaryotic expression method has better activity than commercial recombinant collagen and full-length type III collagen Col3a1. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 It is the SDS-PAGE electrophoresis diagram of recombinant type III collagen CAⅢ-1-MBP and CAⅢ-1-GFP (protein expression); Figure 2 This is a graph showing the results of the experiment on promoting cell migration by recombinant type III collagen CAⅢ-1-MBP, CAⅢ-1-GFP, full-length type III collagen Col3a1 and commercial collagen; Figure 3 This is a statistical experimental result of the scratch recovery rate of repetitive sequence recombinant type III collagen CAⅢ-1-MBP, CAⅢ-1-GFP, full-length type III collagen Col3a1 and commercial collagen; Figure 4 This is a graph showing the results of the cell survival rate experiment of repetitive sequence recombinant type III collagen CAⅢ-1-MBP, CAⅢ-1-GFP, full-length type III collagen Col3a1 and commercial collagen; Figure 5 This is a graph showing the inhibitory effect of recombinant type III collagen CAⅢ-1-MBP and CAⅢ-1-GFP on elastase and collagenase; Figure 6 This is a diagram showing the application effect of the wound dressing prepared using repetitive sequence recombinant type III collagen CAⅢ-1-MBP and CAⅢ-1-GFP as raw materials; Figure 7 This is a graph showing the measurement results of the stratum corneum moisture content of the facial mask products prepared using repetitive sequence recombinant type III collagen CAⅢ-1-MBP and CAⅢ-1-GFP as raw materials; Figure 8 This is a graph showing the results of measuring the skin hemoglobin value of a facial mask product prepared using repetitive sequence recombinant type III collagen CAⅢ-1-MBP and CAⅢ-1-GFP as raw materials. DETAILED DESCRIPTION

[0015] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0016] Example 1: This example provides a prokaryotic expression method of recombinant type III collagen with a repetitive sequence, the specific contents are as follows: The company's type III recombinant collagen repeat sequence protein DNA fragment (SEQ ID NO: 1-3) was codon optimized and then spliced ​​and recombined into the pET28a (+) vector, with restriction sites of BamHI and XhoI. The gene with 6 histidine tags and MBP fusion tags was synthesized and spliced ​​and recombined into the pET28a (+) vector containing the type III recombinant collagen repeat sequence protein DNA fragment, with restriction sites of NcoI and BamHI. The type III recombinant collagen repeat sequence protein expression vector was transformed into Escherichia coli competent cells: BL21 (DE3).

[0017] Single-spot clones were selected from the transformation plate and cultured overnight at 37°C in 50 mL LB medium (containing 100 mg / L kanamycin) (each liter of medium contains 10 g peptone, 5 g yeast powder and 5 g NaCl). Transferred to 1.8 liters LB medium (containing 100 mg / L kanamycin) at a ratio of 1:36. Cultured at 37°C until OD600 was 0.4-0.6, and 0.5 mM IPTG was added to induce protein expression at 16°C.

[0018] The bacterial liquid was centrifuged at 7000rpm for 10min to collect the bacteria. The bacterial precipitate was resuspended in 10mM imidazole PBS (pH7.5) solution, the bacteria were broken under high pressure, and centrifuged at 4°C, 11000rpm for 15min. The supernatant was taken and incubated with a balanced 5mL nickel column (Thermo Fisher: 88221). Gradient elution was performed with 10mM PBS solution (pH7.5) to wash away the impurities, and gradient elution was performed with 30mM, 50mM, 100mM, 200mM and 300mM imidazole PBS solutions (pH7.5) respectively. The protein was detected by SDS-PAGE, and ultrafiltration was used to concentrate and remove imidazole. The obtained type III recombinant collagen repeat sequence protein CAⅢ-1-MBP was subjected to SDS-PAGE detection, and the results are as follows: Figure 1 As shown by Figure 1 It can be seen that CAⅢ-1-MBP has obvious bands, while CAⅢ-1 (prokaryotic) without a fusion tag has basically no expression, which indicates that the fusion tag can significantly increase the expression level of type III recombinant collagen repeat sequence protein in the prokaryotic system.

[0019] Example 2: This example provides a method for eukaryotic expression of recombinant type III collagen with a repetitive sequence, the specific contents of which are as follows: Construction of expression vector: The type III recombinant collagen repeat sequence protein DNA fragment (SEQ ID NO: 1-3) synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. was codon optimized and then spliced ​​and recombined into the pcDNA3.1(+) vector, with restriction sites of BamHI and XhoI. The gene with 8 histidine tags and GFP fusion tags was synthesized and spliced ​​and recombined into the pcDNA3.1(+) vector containing the type III recombinant collagen repeat sequence protein DNA fragment, with restriction sites of NheI and BamHI.

[0020] Plasmid extraction: Transform the constructed plasmid vector into E. coli DH5α, place it in an ice bath, heat shock, and ice bath, then add 1mL LB liquid culture medium to activate it at 37°C, 200rpm for 40min, apply LB solid culture medium plate (0.1mg / mL ampicillin sodium), incubate it inverted at 37°C for 12h, pick a single colony and add it to 100mL LB liquid culture medium (0.1mg / mL ampicillin sodium), and culture it at 37°C, 200rpm for 12h. Use the plasmid large-scale extraction kit (Biyuntian: D0025-3) to extract the plasmid, and use the Nanodrop instrument to detect the concentration of the vector. It can be stored at 4°C for one month, and it is recommended to be used up within one week.

[0021] Protein expression: Expi293F cells were pre-suspended and cultured in a shaker at 37°C, 110 rpm, 80% humidity, and 7% carbon dioxide. The culture medium used was serum-free culture medium (Gitex: HE000-N012). When the cell density reached 3.0×10 6 / mL, and the viable cell rate is >95%, perform plasmid transfection. Use the plasmid transfection kit (Biyuntian: C0518) to transfect Expi293F cells (taking 40mL as an example): 80μL of transfection reagent is added to 1mL of cell culture medium, and gently mixed to obtain solution A; 40μg of plasmid is added to 1mL of cell culture medium, and gently mixed to obtain solution B. Solution B is added to solution A, gently mixed, and allowed to stand for 15 minutes. Add the mixture of A and B to 40mL of suspended Expi293F cells, continue to culture for 7 days, detect the viable cell rate every day, and harvest the cells when the viable cell rate is lower than 50%.

[0022] Protein purification: The cell suspension was centrifuged at 4°C, 5000rpm for 5min, the supernatant culture medium was taken, incubated with 5mL nickel column (Thermo Fisher: 88221), eluted by gravity, washed with 50mL PBS solution containing 10mM imidazole, eluted with 300mM imidazole PBS solution (pH7.5), SDS-PAGE protein detection, the PBS eluate containing the target protein and good purity was combined, ultrafiltration tube (Millipore, UFC9010) was ultrafiltered for concentration and removal of imidazole. The obtained type III recombinant collagen repeat sequence protein CAⅢ-1-GFP was detected by SDS-PAGE, and the results are as follows: Figure 1 As shown by Figure 1 It can be seen that CAⅢ-1-GFP has obvious bands, while CAⅢ-1 (eukaryotic) without a fusion tag has basically no expression, which indicates that the fusion tag can significantly increase the expression level of type III recombinant collagen repeat sequence protein in the eukaryotic system.

[0023] Experimental Example 1: Determination of the activity of recombinant type III collagen in promoting cell migration: Mark the back of the 12-well plate with a ruler and draw horizontal lines evenly, every 0.5 cm, and draw 3 lines in each well. Add about 5×10 4 BALB / c 3T3 cells were cultured overnight to allow the cells to adhere to the wall. The next day, the cells were scratched with a ruler using a sterile pipette tip, washed three times with PBS, the scratched cells and the remaining DMEM medium with serum were removed, serum-free DMEM medium was added, and the control group, CAⅢ-1-MBP, CAⅢ-1-GFP, full-length type III collagen Col3a1 and commercial recombinant collagen experimental groups (0.1 mg / mL) were set up, and the cells were returned to the incubator. Samples were taken at 0, 6, and 12 hours and photographed. The results are shown in the figure. Figure 2 As shown by Figure 2 As far as we know, CAⅢ-1-MBP and CAⅢ-1-GFP showed better cell migration effect. Three replicate wells were set in each group, and 5 points were selected in each well for statistics. The results of the scratch recovery rate statistical experiment are shown in Figure 3 As shown by Figure 3 It can be seen that after 12 hours of action, the four experimental groups of 0.1 mg / mL CAⅢ-1-MBP (scratch recovery rate of 33%), CAⅢ-1-GFP (scratch recovery rate of 55%), full-length type III collagen Col3a1 (scratch recovery rate of 45%) and commercial recombinant collagen (scratch recovery rate of 37%) showed obvious activity in promoting cell migration (with statistical difference) compared with the control group (scratch recovery rate of 30%). The protein CAⅢ-1-GFP expressed by eukaryotic cells has the best activity in promoting cell migration, and is significantly better than the commercial recombinant collagen.

[0024] Experimental Example 2: Cytotoxicity Evaluation of Repeat Sequence Recombinant Type III Collagen: Take HeLa cells that have grown to 70%-80% of the bottom area of ​​the culture flask, digest them with 0.25% trypsin, and use complete culture medium to make the cell density of 1×10 5 / mL cell suspension. Take 100mL of cell suspension and inoculate it in a 96-well culture plate and place it in an incubator for culture. After 24 hours of cell culture, aspirate the complete culture medium. CAⅢ-1-MBP, CAⅢ-1-GFP, full-length type III collagen Col3a1 and commercial recombinant collagen diluted with high-glucose DMEM medium at a concentration of 0.1mg / mL were added to the experimental group. Any collagen involved three parallel groups. The control group was cells cultured in DMEM medium, and the blank group was DMEM medium without cells and continued to be cultured in the incubator for 24 hours. Add 10μL of CCK-8 reagent to each group, incubate in the cell culture incubator for 3 hours, use an enzyme reader to detect the absorbance value OD at 450nm, and calculate the cell survival rate according to the absorbance mean of each group. Cell survival rate = (experimental group-blank group) / (control group-blank group)×100%, unit: %; The results are as follows Figure 4 As shown by Figure 4 It can be seen that at a concentration of 0.1 mg / mL, the cell survival rate was not affected, proving that CAⅢ-1-MBP, CAⅢ-1-GFP, full-length type III collagen Col3a1 and commercial recombinant collagen all have high safety.

[0025] Experimental Example 3: Inhibitory effect of recombinant type III collagen with repeated sequences on elastase: Porcine pancreatic elastase (EC 3.4.21.36) was dissolved and prepared into a stock solution of 3.33 mg / mL in sterile water. The substrate N-succinyl-alanine-alanine-p-nitroaniline was dissolved in a 1.6 mM buffer, which was a 0.2 mM Tris-HCL buffer (pH 8.0). CAⅢ-1-MBP (or CAⅢ-1-GFP) was incubated with the enzyme in the buffer for 15 min, and then the substrate was added. The total reaction volume was 250 μL, containing 0.8 mM substrate, 1 μg / mL elastase, and 25 μL of CAⅢ-1-MBP obtained in Example 1 (or CAⅢ-1-GFP obtained in Example 2). Water was used as a negative control, and the positive control was 0.114 mg / mL epigallocatechin gallate. The absorbance value was detected at 390 nm, and the elastase inhibition rate = ((negative control group-sample group) / negative control group) × 100%.

[0026] Experimental Example 4: Inhibitory effect of recombinant type III collagen with repeated sequences on collagenase: CAⅢ-1-MBP (or CAⅢ-1-GFP) and enzyme were incubated in buffer for 15 minutes, and then hydroxyproline-malonic acid-tyrosine tripeptide (substrate) was added. The total reaction volume was 150 μL, containing 0.8 mM hydroxyproline-malonic acid-tyrosine tripeptide, 0.1 unit of Clostridium histolyticum collagenase (EC.3.4.23.3), and 25 μL of CAⅢ-1-MBP obtained in Example 1 (or CAⅢ-1-GFP obtained in Example 2), and buffer was used for preparation. Water was used as a negative control, and 0.114 mg / mL of epigallocatechin gallate was used as a positive control. The absorbance value was detected at 335 nm, and the collagenase activity inhibition rate = ((negative control group-sample group) / negative control group) × 100%.

[0027] The inhibitory effects of recombinant type III collagen CAⅢ-1-MBP and CAⅢ-1-GFP on elastase and collagenase are as follows Figure 5 As shown by Figure 5 It can be seen that CAⅢ-1-MBP and CAⅢ-1-GFP have a high inhibitory effect on elastase and collagenase, and can restore skin elasticity and slow down skin aging.

[0028] Application Example 1: Repeated sequence recombinant type III collagen is used as raw material for the preparation of wound dressings: 1) Preparation of wound dressing: 1.5 g chitosan was dissolved in 100 mL of HCL solution with a pH of 2.5, and stirred at room temperature until uniformly dispersed to obtain chitosan solution. 3 g of EDC (1-ethyl (3-dimethylaminopropyl) carbodiimide) and 945 mg of dihydrocaffeic acid were dissolved in 60 mL of anhydrous ethanol, and the chitosan solution was slowly added to react at room temperature for 24 h. After completion, the supernatant was dialyzed in HCL solution (pH = 7.4) for 3 days. The material in the dialysis bag was finally freeze-dried and stored at -20 ° C for standby use, which was recorded as the matrix. Taking CAⅢ-1-MBP as an example, phosphonate, matrix, and CAⅢ-1-MBP were weighed and added to ultrapure water. By mass percentage, the phosphonate was 0.5%, the matrix was 25%, and the CAⅢ-1-MBP was 3%. After stirring until uniformly dispersed, CAⅢ-1-MBP wound dressing was obtained. The preparation of CAⅢ-1-GFP wound dressing was the same as above.

[0029] 2) Wound dressing effect verification: The obtained CAⅢ-1-MBP wound dressing and CAⅢ-1-GFP wound dressing were subjected to cytotoxicity evaluation, cell migration detection, antibacterial detection and animal experiments. The following shows the relevant content of animal experiments: The experiment was conducted using 6-8 week old male BALB / c mice, all of which were randomly divided into three groups (blank control group, CAⅢ-1-MBP wound dressing and CAⅢ-1-GFP wound dressing) and adapted to the feeding environment for 1 week. Then, the mice were anesthetized by intraperitoneal injection of chloral hydrate, and a 10 mm diameter full skin defect wound was created on the back. CAⅢ-1-MBP wound dressing and CAⅢ-1-GFP wound dressing were injected into the wound by syringe, respectively. After 15 mW blue light irradiation for 45 seconds, the wound was disinfected and bandaged with iodine. The wound was photographed at 0, 4, 8, and 12 days, and the wound area was measured using Image J software.

[0030] The results are as follows Figure 6 As shown by Figure 6 It can be seen that compared with the blank group, both CAⅢ-1-MBP wound dressing and CAⅢ-1-GFP wound dressing showed better wound recovery effects, among which CAⅢ-1-MBP wound dressing performed slightly better, and the wounds of mice were basically healed after 12 days.

[0031] Application Example 2: Repeating sequence recombinant type III collagen is used as raw material for the preparation of skin care products: 1) Preparation of skin care products: Specifically, it is prepared into a facial mask product, which contains the following ingredients in parts by weight: 3 parts of CAⅢ-1-MBP solution, 0.2 parts of hydroxyethyl cellulose, 0.1 parts of carbomer, 0.08 parts of sodium methyl paraben, 0.05 parts of sodium propyl paraben, 0.1 parts of phenoxyethanol, 3 parts of glycerol, 2 parts of γ-oryzanol, 0.2 parts of senna acid, 1 part of buddleja extract, CAⅢ-1-MBP solution is a 10g / L solution of CAⅢ-1-MBP obtained in Example 1, and the above-mentioned weight parts of ingredients are weighed and mixed until uniformly dispersed, and then loaded onto a non-woven fabric to obtain a facial mask product, which is recorded as a CAⅢ-1-MBP facial mask product. Correspondingly, the preparation of the CAⅢ-1-GFP facial mask product is the same as above, and a facial mask product without CAⅢ-1-MBP or CAⅢ-1-GFP is prepared for control.

[0032] 2) Verification of the effectiveness of skin care products: The obtained CAⅢ-1-MBP mask products and CAⅢ-1-GFP mask products were subjected to antibacterial and antimicrobial effect testing, skin allergy testing and efficacy testing. The efficacy test was conducted after recruiting volunteers and was conducted at Hangzhou Saiwei Quality Technology Service Co., Ltd. The test indicators involved stratum corneum moisture content, transepidermal water loss value, skin hemoglobin value, and redness area a* value.

[0033] The results are as follows Figure 7 , Figure 8 As shown by Figure 7 , Figure 8It can be seen that facial mask products containing recombinant type III collagen CAⅢ-1-MBP and CAⅢ-1-GFP with repeated sequences have better hydrating, anti-redness and repairing effects.

[0034] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope of the technical solution of the present invention.

Claims

1. A repetitive sequence recombinant type III collagen, characterized in that: The repetitive sequence recombinant type III collagen includes CAⅢ-1, CAⅢ-2, and CAⅢ-3, and can be prepared by a prokaryotic expression method or a eukaryotic expression method; Repeat sequence recombinant type III collagen CAⅢ-1, composed of 387 amino acids, the basic repeating unit is GAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAP, from the peptide segment 438~512 of human type III collagen, 5 repeats, and the C-terminus contains the type III collagen 1189-1200 fragment; Repeat sequence recombinant type III collagen CAⅢ-2, composed of 387 amino acids, the basic repeating unit is PPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQG, a peptide segment from human type III collagen 565-639, 5 repeats, and the C-terminus contains the type III collagen 1189-1200 fragment; The repetitive sequence recombinant type III collagen CAⅢ-3 is composed of 327 amino acids. The basic repeating unit is PPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPG, which is a peptide segment from 940 to 1002 of human collagen III, with 5 repeats and the C-terminus contains the type III collagen 1189-1200 fragment.

2. The repetitive sequence recombinant type III collagen according to claim 1, characterized in that: The invention relates to a prokaryotic expression method of recombinant type III collagen of a repetitive sequence, wherein the expression vector is pET28a(+), and the expression is carried out in an Escherichia coli expression strain, wherein the expression strains include: BL21(DE3), BL21(DE3)pLysS, BL21Star(DE3), BL21Star(DE3)pLysS, Rosetta(DE3), Rosetta(DE3)pLysS, OrigamiB(DE3), OrigamiB(DE3)pLysS, SHuffle T7, BLR(DE3), BLR(DE3)pLysS, B834(DE3), and B834(DE3)pLysS.

3. The repetitive sequence recombinant type III collagen according to claim 2, characterized in that: The eukaryotic expression method of recombinant type III collagen with a repetitive sequence is carried out using an expression vector of pcDNA3.1(+) or pFastBac1, and the eukaryotic cells for expression include Expi293, ExpiCHO, and Expi sf9.

4. The repetitive sequence recombinant type III collagen according to claim 3, characterized in that: The expression vector contains a histidine tag and a fusion tag. The histidine tag is 6-8 histidines, and the fusion tag is MBP or GFP.

5. An application of the repetitive sequence recombinant type III collagen according to any one of claims 1 to 4, characterized in that: Repeated sequence recombinant type III collagen CAⅢ-1, CAⅢ-2, and CAⅢ-3 are used as raw materials for the preparation of wound dressings and skin care products.

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