A recombinant human collagen xv and preparation method and application thereof

Abstract

The application relates to the technical field of bioengineering, and particularly discloses a recombinant human collagen XV and a preparation method and application thereof. The amino acid sequence of the recombinant human collagen XV is shown as SEQ ID NO. 1. The recombinant human collagen XV is derived from human collagen, and has no rejection and sensitization risks. Experiments show that the protein has good effects of promoting cell proliferation and promoting cell migration, and has good biocompatibility, and can be applied as a safe and effective biomedical material.

Description

Technical Field

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

[0002] Collagen is one of the most abundant proteins in the human body, mainly found in tissues such as skin, bones, tendons, ligaments, and blood vessels. It plays a crucial role in maintaining tissue structure and function, accounting for approximately 30% of the total protein in the human body. Due to its excellent biological functions, biocompatibility, and biodegradability, collagen has become one of the most promising protein materials in fields such as biomaterials and regenerative medicine. Currently, most commercially available collagen is obtained through extraction from animal tissues, but these traditional extraction methods have many problems. On the one hand, the extraction process often alters the collagen structure, resulting in differences from its natural state in the body; on the other hand, there are also drawbacks such as strong immune rejection, inconsistent product batch quality, and unstable biological efficacy. These factors greatly limit the application range of collagen materials. With the rapid development of modern biotechnology such as genetic engineering, protein engineering, and synthetic biology, recombinant protein expression technology has become an important option. As a substitute for natural animal tissue collagen, recombinant collagen shows great potential for widespread application in the fields of biomaterials and biomedicine due to its excellent biocompatibility, low immunogenicity, processability, and controllable biological efficacy.

[0003] XV-type collagen is widely distributed in various tissues, with the strongest expression in the basement membrane region. Its main function is to adhere the basement membrane to the underlying connective tissue matrix. This characteristic makes XV-type collagen play a crucial role in tissue repair, particularly in the repair of damage to skin, muscles, and blood vessels. However, despite some progress in the study of the structure and function of XV-type collagen, its content in animals is low, making it difficult to extract a single component. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a recombinant human XV type collagen, its preparation method, and its applications.

[0005] The present invention provides a recombinant human XV type collagen, wherein the amino acid sequence of the recombinant human XV type collagen is shown in SEQ ID NO.1.

[0006] The recombinant human XV collagen of this invention is based on the original amino acid sequence of human XV collagen. Key fragments with high biological activity and strong structural stability were retained through screening, resulting in a novel recombinant human XV collagen. This recombinant human XV collagen has the full-length chain sequence at the C-terminus and N-terminus of the collagen coding region removed, effectively avoiding a series of antigen-immune reactions. Its theoretical molecular weight is 37.30 kDa, which is moderate and easy to prepare. This invention also demonstrated efficient secretory expression of recombinant human XV collagen in a fermenter, achieving a yield of 2.36 g / L in a 5L fermenter.

[0007] The present invention also provides a gene encoding the above-mentioned recombinant human XV type collagen, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] An expression vector containing the gene.

[0009] A host cell comprising the expression vector described above.

[0010] The host cell is either Escherichia coli or Pichia pastoris.

[0011] A method for preparing recombinant human XV type collagen includes: culturing host cells containing the gene encoding the recombinant human XV type collagen, and causing them to express the recombinant human XV type collagen.

[0012] Preferably, the host cells are induced to express the recombinant human XV type collagen using an inducer.

[0013] Preferably, the inducing agent is methanol.

[0014] Preferably, purification is also included.

[0015] Preferably, the purification is performed using one or more of the following methods: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation.

[0016] The recombinant human XV type collagen is used in the preparation of tissue repair products, wherein the tissue includes any one of skin, muscle, and blood vessels.

[0017] The materials include any one of skin care products, health products, tissue engineering materials, and medical devices.

[0018] The recombinant human XV type collagen of the present invention is used to promote cell proliferation.

[0019] The recombinant human XV type collagen of the present invention is used to promote cell migration.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The recombinant human XV type collagen of the present invention is based on the original amino acid sequence of human XV type collagen. After screening, key fragments with high biological activity and strong structural stability were retained, resulting in a novel recombinant human XV type collagen.

[0022] (2) The recombinant human XV collagen of the present invention has removed the full-length chain sequence of the C-terminus and N-terminus of the collagen coding region, effectively avoiding a series of antigen immune reactions. The theoretical molecular weight of this protein is 37.30 kDa, which is moderate and easy to prepare.

[0023] (3) The present invention also carried out small-scale expression in a fermenter to achieve efficient secretory expression of recombinant human XV type collagen. The yield of 5L fermenter can reach 2.36g / L.

[0024] (4) The recombinant human XV type collagen of the present invention has good cell proliferation and cell migration activity and good biocompatibility. Attached Figure Description

[0025] Figure 1 The image shows the SDS-PAGE analysis of the recombinant human XV type collagen XV-401 produced by shake-flask fermentation according to the present invention. In the image, M is the protein molecular weight standard, and 1, 2, 3 and 4 are the results of shake-flask fermentation of recombinant human XV type collagen at 0h, 24h, 48h and 72h, respectively.

[0026] Figure 2 The image shows the SDS-PAGE analysis of recombinant human XV type collagen XV-401 in a 5L fermenter according to the present invention, where M is the protein molecular weight standard and I is the result of fermentation of recombinant human XV type collagen in the fermenter.

[0027] Figure 3 The image shows an SDS-PAGE analysis of the recombinant human XV type collagen XV-401 purified by ion exchange chromatography according to the present invention. In the image, M is the protein molecular weight standard, 1 is the result of the protein loading solution, 2 is the result of the protein washing solution, and 3 is the result of the protein elution solution.

[0028] Figure 4 The bar chart shows the cell proliferation of recombinant human XV type collagen XV-401 as described in this invention. From left to right, the control group (without treatment of human umbilical vein endothelial cells with recombinant human XV type collagen XV-401), the experimental group with a drug concentration of 0.5 mg / ml, the experimental group with a drug concentration of 1 mg / ml, the experimental group with a drug concentration of 3 mg / ml, and the experimental group with a drug concentration of 5 mg / ml.

[0029] Figure 5The figures show the cell migration of recombinant human XV type collagen XV-401 as described in this invention. Figures (A), (B), and (C) show the migration of human umbilical vein endothelial cells and recombinant human XV type collagen XV-401 after co-culturing for 0 h, 4 h, and 8 h, respectively. Detailed Implementation

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0031] The amino acid sequence of the recombinant human XV type collagen provided by the present invention is shown in SEQ ID NO.1. Because it contains a total of 401 amino acid sequences, it is named recombinant human XV type collagen XV-401.

[0032] SEQ ID NO.1: GMKGQAGPKGEKGDAGEEGPPGPPGPPGLPGIPGKPGTDVFMGPPGSPGEDGPAGEPGPPGPEGQPGVDGATGLPGMKGEKGARGPNGSVGEKGDPGNRGLPGPPGKKGQAGPPGVMGPPGPPGPPGPPGPGGLKGEKGDRGPKGERGMDGASIVGPPGPRGPPGHIGPPGPDGLPGLPGFPGPRGPKGDTGLPGFPG LKGEQGEKGEPGAIGKKGEPGMHGAPGPMGPKGPPGHKGEFGLPGRPGRPGLNGLKGTKGDPGVIMQGPPGLPGPPGPPGPPGAVGVKGEKGSWGLPGSKGE KGDQGAQGPPGPPGENGDKGFKGEKGEKGDIGPPGLPGNPGPAGQKGETVVGPQGPPGAPGLPGPPGFGRPGDPGPPGPPGPPGPPGPPGPPGQPGLPGSR.

[0033]

[0034] Example 1

[0035] Preparation of recombinant human XV type collagen XV-401 in Pichia pastoris expression system

[0036] Experimental methods

[0037] 1. Preparation of shuttle plasmid and Pichia pastoris expression strain

[0038] According to the amino acid sequence shown in SEQ ID No. 1, codon optimization was performed on the Pichia pastoris expression system to obtain the target gene sequence of recombinant human XV type collagen XV-401. The obtained target gene sequence was synthesized by Qingke Biotechnology Co., Ltd., and the synthesized gene was ligated into the pPIC9K vector to obtain a recombinant plasmid. The recombinant plasmid was transformed into Pichia pastoris host cells to obtain the TOP10 strains containing pPIC9K-XV-401. Then, the recombinant plasmid was extracted, linearized, electroporated, and positive transformants were screened. The specific steps are as follows:

[0039] (1) Extraction of recombinant plasmids

[0040] Plasmid extraction was performed using a plasmid extraction kit. RNase A, solution P1, solution P2, and solution P4 are all reagents in the plasmid extraction kit, which was purchased from Tiangen Biotech (Beijing) Co., Ltd., model DP117.

[0041] 1) Take 10 μl of the TOP10 strain containing pPIC9K-XV-401 and inoculate it into 100 ml of liquid LB medium, and incubate overnight.

[0042] 2) Add 100 ml (adjust the amount according to the concentration of the cultured bacteria; 200 ml is recommended for low copy numbers) of the overnight culture to a centrifuge tube. Centrifuge at 8000 rpm for 3 minutes at room temperature to collect the bacteria, and remove as much supernatant as possible. Note: If there is a large amount of bacterial culture, you can centrifuge several times to collect the bacterial precipitate into one centrifuge tube. The amount of bacterial culture should be enough to allow for sufficient lysis; too much bacterial culture will lead to incomplete lysis and reduce the plasmid extraction efficiency.

[0043] 3) Add 8 ml of RNase A solution P1 to the centrifuge tube containing the bacterial precipitate, and thoroughly resuspend the precipitate using a pipette. Note: It is essential to thoroughly resuspend the bacterial precipitate. Any unmixed bacterial clumps will affect the lysis effect, resulting in lower extraction yield and purity.

[0044] 4) Add 8 ml of solution P2 to the centrifuge tube, and immediately and gently invert it 6-8 times to ensure complete lysis of the bacteria. Incubate at room temperature for 5 minutes. Note: Mix gently, do not shake violently to avoid contaminating the genomic DNA. At this point, the bacterial culture should become clear and viscous. If it does not become clear, it may be due to an excessive number of bacteria and incomplete lysis; the amount of bacteria should be reduced.

[0045] 5) Add 8 ml of solution P4 to the centrifuge tube, and immediately and gently invert it 6-8 times to mix thoroughly until a white, dispersed flocculent precipitate appears. Then, let it stand at room temperature for about 10 minutes, centrifuge at 8000 rpm for 5-10 minutes to allow the white precipitate to settle to the bottom of the tube (centrifugation time can be increased appropriately). Carefully pour all the solution into filter CS1 (avoid pouring in a large amount of precipitate that could clog the filter), and slowly push the push handle to filter. Collect the filtrate in a clean, self-prepared 50 ml centrifuge tube. Note: Mix immediately after adding solution P4 to avoid localized precipitation. A white precipitate in the solution poured into filter CS1 after centrifugation will not affect filtration. If the bacterial count is too high (>100 ml), it is recommended to extend the centrifugation time to 20 minutes.

[0046] 6) Column equilibration step: Add 2.5 ml of equilibration solution BL to the adsorption column CP6 (place the adsorption column in a 50 ml collection tube), centrifuge at 8000 rpm for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube (the column treated with equilibration solution is best used immediately).

[0047] 7) Add 0.3 times the volume of isopropanol to the filtrate (adding too much isopropanol can easily lead to RNA contamination), mix thoroughly by inverting, and then transfer to the CP6 adsorption column (place the adsorption column in a 50ml collection tube). Note: Some filtrate will be lost after filtration; add different volumes of isopropanol depending on the amount lost. The maximum volume of the CP6 adsorption column is 15ml, so it needs to be filtered twice. In some cases, the centrifuge rotor tilt angle may be large; in this case, it is recommended that the volume of CP6 solution added to the adsorption column not exceed 10ml to prevent leakage.

[0048] 8) Centrifuge at 8000 rpm for 2 minutes at room temperature, discard the waste liquid in the collection tube, and put the CP6 adsorption column back into the collection tube. Note: Pass the solution obtained in step 7 through the column twice, following the above conditions each time.

[0049] 9) Add 10 ml of anhydrous ethanol wash solution PW to the adsorption column CP6, centrifuge at 8000 rpm for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0050] 10) Repeat step 9).

[0051] 11) Add 3 ml of anhydrous ethanol to the adsorption column CP6, centrifuge at 8000 rpm for 2 min at room temperature, and discard the waste liquid.

[0052] 12) Place the adsorption column CP6 back into the collection tube and centrifuge at 8000 rpm for 5 min at room temperature to remove the residual washing solution in the adsorption column.

[0053] 13) Open the CP6 adsorption column and place it at room temperature for several minutes to thoroughly dry any residual rinsing solution in the adsorption material.

[0054] 14) Place the CP6 adsorption column in a clean 50ml collection tube, add 1-2ml of elution buffer TB to the middle of the adsorption membrane, incubate at room temperature for 5min, and then centrifuge at 8000rpm for 2min at room temperature.

[0055] 15) Add the elution buffer from the 50ml centrifuge tube back into the adsorption membrane from the previous step and centrifuge again at 8000rpm for 2min at room temperature.

[0056] 16) Transfer all the elution buffer from the 50 ml centrifuge tube into a clean 1.5 ml centrifuge tube and store at -20°C.

[0057] (2) Plasmid linearization

[0058] 1) Linearized system

[0059] Table 1. Plasmid linearization system

[0060]

[0061] 2) Linearization steps

[0062] a. Prepare all reagents into 1.5 ml centrifuge tubes according to the linearization system, and then place the centrifuge tubes in a 37°C water bath for 1 h.

[0063] b. Place the centrifuge tubes in a 70°C water bath and react for 15 minutes to terminate the reaction.

[0064] c. Add 2.5 × V volume of anhydrous ethanol and 3.5 × 0.05 × V volume of 5M NaCl to the centrifuge tube.

[0065] d. Place the centrifuge tubes at -20℃ for 20 minutes.

[0066] e. Centrifuge at 12000 rpm for 10 min at room temperature, collect the supernatant, air dry, and add 12 μl of enzyme-free sterile water for recovery.

[0067] (3) Electro-rotation

[0068] 1) Remove the competent Pichia pastoris cells from the -80℃ freezer and place them on ice to thaw.

[0069] 2) Take out the recovered linearized DNA fragment (about 10 μl), add it to competent cells, gently pipette to mix, and transfer to an electroporation cup in an ice bath.

[0070] 3) Use an electric shock device to deliver electric shocks. Set the parameters as follows: voltage 1.5 kV, resistance 200Ω, capacitance 25 μF, and shock time 5ms (two shocks).

[0071] 4) Immediately after the electric shock, add 1M sorbitol in an ice bath, transfer to a 1.5ml centrifuge tube, and incubate at 30℃ for 1h.

[0072] 5) Centrifuge at 6000 rpm for 5 min at room temperature and collect part of the supernatant.

[0073] 6) Resuspend the bacterial pellet in the remaining supernatant, take 200 μl and spread it on an MD plate, and incubate at 30°C for 3-5 days.

[0074] (4) Screening of positive transformants

[0075] Positive transformants grown on MD plates were selected and screened for high copy numbers on YPD plates containing 6 mg / ml, 4 mg / ml and 2 mg / ml of G418 resistance, respectively.

[0076] 2. Permeabilization expression of the target protein

[0077] (1) Shake-flask induced expression

[0078] 1) Pick positive transformants from 6 mg / ml G418 plates, inoculate them into 50 ml BMGY medium (1% yeast extract, 10% PPB, 1.34% YNB, 2% peptone and 1% glycerol), and then incubate on a shaker (28℃, 220 rpm) for 24 h.

[0079] 2) Measure the OD of the BMGY shake flask 600 Value (OD) 600 =10~13), transfer a portion of the bacterial culture to BMMY liquid medium (1% yeast extract, 10% PPB, 1.34% YNB, 2% peptone and 1% methanol) to increase its OD. 600 The value is 1, and then it is placed in a shaker (30℃, 220rpm) for 72h.

[0080] 3) Add 1% methanol to the culture medium every 24 hours and take 1 ml of the sample into a 1.5 ml centrifuge tube. Centrifuge the sample at 12,000 rpm for 5 minutes at room temperature and collect the supernatant. Analyze the expression level of the target protein and the optimal collection time of the bacterial culture. The sampling time is generally 0 h, 24 h, 48 h, and 72 h.

[0081] 4) Perform SDS-PAGE protein electrophoresis on the supernatant of the fermentation broth collected at different time intervals.

[0082] The results of shake-flask permeabilization expression of the target protein are as follows: Figure 1 As shown, M is the protein molecular weight standard, and 1, 2, 3 and 4 are the results of shake-flask fermentation of recombinant human XV type collagen at 0h, 24h, 48h and 72h, respectively.

[0083] (2) Small-scale expression in fermenter

[0084] 1) Select single colonies of the constructed Pichia pastoris expression strain TOP10 and add them to 50ml of BMGY liquid medium (1% yeast extract, 10% PPB, 1.34% YNB, 2% peptone and 1% glycerol), and incubate on a shaker (30℃, 220rpm) for 24h (OD). 600 Activation is performed using 10~20 (units unspecified).

[0085] 2) Transfer 1% inoculum to a 500ml Erlenmeyer flask (containing 100ml BMGY culture medium) and incubate on a shaker (30℃, 220rpm) for 24 hours (OD). 600 =8~10), as the seed liquid for the upper tank.

[0086] 3) Prepare a high-density fermentation medium (3L). The medium composition is as follows: 120g glycerol, 27.3g K2SO4, 1.38g CaSO4·2H2O, 6.3g KOH, 22.5g MgSO4·7H2O, 40.2ml phosphoric acid, 2ml defoamer, 50g glycerol and 2500ml water.

[0087] 4) Add 3L of high-density fermentation medium to a 5L fermenter and sterilize at 121℃ for 20min. After cooling to 30℃, add 12ml of PTM1 (CuSO4-5H2O 6.0g / L, NaI 0.088g / L, MnSO4-H2O 3.0g / L, Na2MoO4-2H2O 0.2g / L, H3BO3 0.02g / L, CoCl2-6H2O 0.5g / L, ZnCl2 20.0g / L, FeSO4-7H2O 65.0g / L, Biotin 0.2g / L, concentrated H2SO4 5.0ml and H2O). Add an appropriate amount of ammonia to adjust the pH to 5. Add the seed culture prepared in step 2) to the fermenter by flame inoculation for fermentation culture.

[0088] 5) Cultivate to OD 600 When the concentration of glycerol reaches 150-200, stop glycerol feeding. Measure the OD value every 30 minutes after stopping the feeding. 600 The value until OD 600 The value remained basically unchanged, and periodic methanol feeding was performed to maintain dissolved oxygen at around 10%.

[0089] 6) Sampling and OD are performed every 4 hours. 600 Value measurement, up to OD 600 Once the bacteria begin to descend, they can be harvested.

[0090] The results of the small-scale expression of the target protein in a fermenter are as follows: Figure 2 As shown, M represents the protein molecular weight standard, used to indicate the molecular weight corresponding to the protein band. 1 represents the result of loading the container.

[0091] 3. Purification of recombinant human XV type collagen XV-401

[0092] (1) Prepare sample loading buffer A (1L) (NaH2PO4 2.4696g, Na2HPO4 0.2985g) and elution buffer B (1L) (NaH2PO4 1.656g, Na2HPO4 1.136g, NaCl 58.44g), and adjust the pH to 6.

[0093] (2) The collected protein fermentation broth was concentrated and its conductivity was reduced by passing it through a 10KD ultrafiltration membrane. The conductivity was reduced to 2000 μs / cm, and solution A was added to keep the conductivity difference between solution A and the concentration within 300 μs / cm. Finally, the pH was adjusted to 6.

[0094] (3) Wash the chromatography column (25 ml Diamond MMC Mustang) with at least 5-10 column volumes of ultrapure water at a constant flow rate of 10 ml / min until the UV baseline is stable.

[0095] (4) Wash the column (25 ml DiamondMMC Mustang) with at least 5-10 column volumes of solution A at a constant flow rate of 10 ml / min until the UV baseline is stable.

[0096] (5) Load the fermentation broth that has been treated with solution A onto the chromatography column at a constant flow rate of 10 ml / min.

[0097] (6) Continue to wash the chromatography column with 100% solution A until the UV absorption baseline returns to a stable state.

[0098] (7) Set up a chromatography system so that the concentration of solution B increases linearly from 0% to 100%. Start collecting the eluent when the UV absorption begins to rise. During elution, collect the eluent in segments according to the peak value monitored by UV.

[0099] (8) Perform SDS-PAGE protein electrophoresis on the collected eluent.

[0100] The purification results of recombinant human XV type collagen XV-401 are as follows: Figure 3 As shown, M represents the protein molecular weight standard, providing a reference for the molecular weight of protein bands in each lane. Figure 1 shows the loading result before purification, revealing multiple protein bands, indicating a complex protein composition and the presence of various proteins in the sample. Figure 2 shows the washing result during purification; most of the other protein bands are present besides the target protein band, indicating that most of the impurities were eluted during the washing process. Figure 3 shows the purification result, displaying the main target protein band, indicating that the target protein was well enriched and purified after ion exchange purification.

[0101] Biological function evaluation of recombinant human XV type collagen XV-401

[0102] 1. Cytotoxicity test

[0103] (1) Human umbilical vein endothelial cells that have grown to 70%-80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin containing EDTA, and then prepared with DMEM / F-12 medium to a cell density of 8×10⁻⁶ cells / year. 4 Cell suspension of cells / ml.

[0104] (2) Take 100 μl of cell suspension and seed it into a 96-well culture plate and place it in an incubator with 37°C and 5% CO2 saturated humidity.

[0105] (3) After 24 h of cell culture, the culture medium was aspirated and different concentrations of recombinant human XV type collagen XV-401 solution (0.5, 1, 3 and 5 mg / ml, with 4 replicates for each concentration) diluted with DMEM / F-12 medium were added to the experimental groups. The control group consisted of cells cultured in DMEM / F-12 medium, and the blank group consisted of DMEM / F-12 medium without cells. The cells were cultured for another 24 h at 37 ℃ and 5% CO2 saturated humidity. After aspirating the culture medium from each group, 110 μl of a mixture of DMEM / F-12 medium and CCK-8 (100 μl medium, 10 μl CCK-8) was added. The cells were then incubated for 1-4 h at 37 ℃ and 5% CO2 saturated humidity in a cell culture incubator. The absorbance (OD) of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. 450 (Value). Cell viability was calculated based on the mean absorbance of each group using the following formula:

[0106] RGR (%) ×100%.

[0107] Experimental results are as follows Figure 4 As shown, compared with the blank control group (100%), the relative cell proliferation rate of the recombinant human XV type collagen XV-401 experimental group prepared in this invention was greater than 100% at low concentrations, and showed a trend of first promoting and then inhibiting with increasing concentration, indicating that the recombinant human XV type collagen prepared in this invention can promote the proliferation of human umbilical vein endothelial cells and its optimal proliferation concentration is 1 mg / ml.

[0108] 2. Cell migration experiment

[0109] (1) Human umbilical vein endothelial cells that have grown to 70%-80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin containing EDTA, and then cultured in DMEM / F-12 medium to a cell density of 2.5×10⁻⁶ cells / year. 5 Cell suspension of cells / ml.

[0110] (2) Use a marker to draw three horizontal lines at the bottom of the well plate with a ruler. Take 2 ml of cell suspension and seed it into a 6-well culture plate and place it in an incubator with 37°C and 5% CO2 saturated humidity.

[0111] (3) After culturing the cells for 24 hours, use a 10 μl pipette tip to vertically align with the well plate using a ruler, gently push downwards to form a longitudinal scratch, aspirate the complete culture medium, rinse 3 times with PBS to remove the scratched cells.

[0112] (4) Add 1 mg / ml of recombinant human XV type collagen XV-401 solution diluted with serum-free DMEM / F-12 medium to the experimental group (set up 2 replicates); the control group is to add an equal amount of serum-free DMEM / F-12 medium without recombinant human XV type collagen XV-401 to the cells.

[0113] (5) Incubate at 37℃ and 5% CO2 saturated humidity for 0h, 4h and 8h respectively. Finally, take a picture under a 10x microscope with the intersection of the horizontal and vertical lines as the core.

[0114] The results are as follows Figure 5 As shown, at 0h ( Figure 5 (As shown in Figure A) The central scratch area was cell-free and cultured in recombinant human XV type collagen XV-401 solution for 4 hours. Figure 5 As shown in Figure B), it was cultured in recombinant human XV type collagen XV-401 solution for 8 hours. Figure 5 (As shown in C) Human umbilical endothelial vein cells migrate to the central scratch area, indicating that recombinant human XV type collagen has the ability to promote the migration of human umbilical vein endothelial cells.

[0115] Based on the above, the recombinant human XV-type collagen XV-401 expressed in this invention has a natural amino acid sequence containing multiple functional sites, uniform molecular weight, good solubility, and excellent cell proliferation and migration properties, making it applicable to various fields. Examples include different forms such as medical dressings, medical spray dressings, medical gel dressings, and medical lyophilized powder dressings, as well as cosmetics and iontophoresis products.

[0116] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0117] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0118] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A recombinant human XV type collagen, characterized in that, The amino acid sequence of the recombinant human XV type collagen is shown in SEQ ID NO.

1.

2. The gene encoding the recombinant human XV type collagen as described in claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. An expression vector comprising the gene of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. The method for preparing recombinant human XV type collagen according to claim 1, characterized in that, This includes: culturing host cells containing the gene encoding the recombinant human XV collagen, and enabling them to express the recombinant human XV collagen.

6. The preparation method according to claim 5, characterized in that, The host cells were induced to express the recombinant human XV type collagen using an inducer.

7. The preparation method according to claim 6, characterized in that, The inducing agent is methanol.

8. The preparation method according to claim 5, characterized in that, It also includes purification.

9. The preparation method according to claim 8, characterized in that, The purification process employs one or more of the following methods: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation.

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