Recombinant collagen type IV with cell migration and proliferation activity and preparation method and application thereof

Abstract

The application provides a recombinant collagen IV with cell migration and proliferation activity and a preparation method and application thereof, and belongs to the technical field of recombinant protein engineering.The recombinant collagen IV with cell migration and proliferation activity is formed by a plurality of repeating units in series, and the amino acid sequence of the repeating unit is shown as SEQ ID NO:1.The preparation method provided by the application comprises the following steps: inserting a nucleotide sequence encoding the amino acid sequence into a carrier to obtain an expression vector; introducing the expression vector into a host cell for culture; inducing the expression of the recombinant collagen IV, and performing separation and purification to obtain the recombinant collagen IV.The recombinant protein obtained by the application has biological activities of promoting cell adhesion, migration and proliferation, and can be widely applied to the fields of biological medicine, tissue engineering and cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein engineering and biotechnology, specifically relating to a recombinant type IV collagen with cell migration and proliferation-promoting activities, its preparation method, and its applications. Background Technology

[0002] Type IV collagen is a core structural protein of the basement membrane. It functions as a support and molecular sieve by forming a network structure, playing a crucial role in maintaining the tissue barrier function of the glomerular basement membrane, alveolar basement membrane, and epidermal-dermal junction of the skin. Due to its excellent biocompatibility and cell adhesion and proliferation-promoting activities, type IV collagen has broad application prospects in tissue engineering, wound repair, and biomedical materials. In particular, its biological activities promoting cell adhesion, migration, and proliferation have attracted significant attention in tissue repair and wound healing.

[0003] Currently, type IV collagen is mainly extracted from animal basement membrane tissue. However, its natural content is extremely low, and it is tightly bound to other basement membrane components, resulting in complex extraction processes, low yields, and difficulties in large-scale production. Using recombinant gene technology to prepare type IV collagen has become an important alternative approach.

[0004] However, full-length type IV collagen has a large molecular weight and complex interchain disulfide bonds, and its correct folding depends on proline hydroxylase-mediated post-translational modifications. Commonly used expression systems such as *E. coli* and *Pichia pastoris* lack sufficient hydroxylation capacity, making soluble expression of the full-length protein almost impossible in these hosts. To address this, researchers have attempted to circumvent these problems by truncating functional fragments of type IV collagen. However, in existing reports, most truncated fragments derived from type IV collagen still fail to achieve detectable levels of secretory expression in recombinant systems; and for some expressible fragments, their cell adhesion, migration, or proliferation-promoting activities are often significantly weakened or even lost. How to obtain a truncated fragment capable of soluble secretory expression in conventional hosts while preserving the cell migration and proliferation-promoting activities of type IV collagen is a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the technical problems existing in the background art, the present invention provides a recombinant type IV collagen with cell migration and proliferation-promoting activities, its preparation method and application. Through systematic screening, a specific truncated fragment of 24 amino acids was identified from the α chain of type IV collagen. This fragment was tandemly repeated as a repeating unit and soluble secreted expression was achieved in Pichia pastoris. At the same time, the obtained recombinant protein has biological activities that promote cell adhesion, migration and proliferation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A recombinant type IV collagen with cell migration and proliferation-promoting activity, wherein the recombinant type IV collagen is formed by multiple repeating units connected in series, and the amino acid sequence of the repeating units is shown in SEQ ID NO:1.

[0007] Further specified, the number of repeating units is 5-30; preferably, the number of repeating units is 10.

[0008] Further specifying, the amino acid sequence of the recombinant type IV collagen is as shown in SEQ ID NO.2.

[0009] A nucleic acid molecule that encodes the aforementioned recombinant type IV collagen.

[0010] An expression vector comprising the aforementioned nucleic acid molecule.

[0011] A host cell comprising the aforementioned recombinant type IV collagen, the aforementioned nucleic acid molecule, or the aforementioned expression vector; the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is *Escherichia coli*; the eukaryotic cell is *Pichia pastoris*.

[0012] A method for preparing recombinant type IV collagen with cell migration and proliferation-promoting activities includes the following steps: Step 1: Insert the nucleotide sequence encoding the above amino acid sequence SEQ ID NO.2 into the vector to obtain the expression vector; Step 2: Introduce the expression vector into the host cells described above and culture them. Step 3: Induce expression of recombinant type IV collagen, then separate and purify it to obtain the recombinant type IV collagen.

[0013] Further specified, the inducing agent used in step 3 is any one of isopropyl-β-D-thiogalactoside, β-galactoside, methanol, or ethanol; more preferably, the inducing agent is methanol; the separation and purification in step 3 is one or a combination of salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation; preferably, the chromatographic chromatography is ion exchange chromatography.

[0014] Application of a recombinant type IV collagen in the preparation of cosmetics, medical devices or tissue engineering materials that promote cell migration and proliferation.

[0015] Further specifying, the medical device includes medical dressings, medical spray dressings, medical gel dressings, or medical lyophilized powder dressings; the tissue engineering material includes cell culture substrates, wound repair materials, or tissue engineering scaffolds.

[0016] The beneficial effects of this invention are: 1. The recombinant type IV collagen of this invention, through systematic screening, identified a specific truncated fragment of 24 amino acids rich in Gly-Pro repeating structures from the α-chain of natural type IV collagen as a repeating unit. This fragment was then tandemly expressed in a Pichia pastoris expression system, achieving soluble secretory expression. The content of recombinant type IV collagen in the fermentation broth reached 1.85 g / L, meeting the requirements for large-scale production. The recombinant type IV collagen obtained by this invention, while achieving soluble secretory expression, still possesses biological activities that promote cell migration and proliferation, and also exhibits good cell adhesion properties, making it suitable for preparing related products that promote tissue repair and regeneration.

[0017] 2. The recombinant type IV collagen of this invention exhibits good biocompatibility and safety. The recombinant type IV collagen of this invention is derived from a truncated fragment of the α-chain of natural type IV collagen, and its amino acid sequence is derived from the natural protein. It possesses good biocompatibility and safety and can be directly applied in fields such as medical devices, tissue engineering, and biomedical materials.

[0018] 3. The preparation method of recombinant type IV collagen of the present invention is simple, easy to operate, and suitable for large-scale industrial production. The recombinant type IV collagen of the present invention can be expressed by fermentation of Pichia pastoris. The target protein in the fermentation broth can be directly separated and purified without the need for a complicated in vitro refolding process. The separation and purification steps are simple, the production cost is controllable, and it is suitable for large-scale industrial production. Attached Figure Description

[0019] Figure 1 This is an SDS-PAGE analysis diagram of the recombinant type IV collagen shake flask test of the present invention; Figure 2 This is an SDS-PAGE analysis image of the recombinant type IV collagen purified from a 5L fermenter according to the present invention; Figure 3 This is a mass spectrometry diagram of the recombinant type IV collagen of the present invention. Figure 4 This is the ultraviolet spectrum of the recombinant type IV collagen of this invention; Figure 5 This is the circular dichroism (CD) spectrum of the recombinant type IV collagen of this invention; Figure 6 This is the Fourier transform infrared (FTIR) spectrum of the recombinant type IV collagen of this invention; Figure 7 This is a bar chart showing the cytotoxicity and cell proliferation activity of the recombinant type IV collagen of this invention. Figure 8 The images show cell migration and scratch healing of the recombinant type IV collagen of this invention (0 h, 24 h). Detailed Implementation

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of methods consistent with some aspects of the invention as detailed in the appended claims.

[0022] The terminology used in this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms “a,” “the,” and “the” used in this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0023] In the description of this invention, it should be understood that the numerical labels before the steps do not indicate the order in which the steps are performed, but are only used to facilitate the description of this application and to distinguish each step, and therefore should not be construed as a limitation of this application.

[0024] The research and development concept of this invention is as follows: a truncated protein segment from the α chain of natural type IV collagen (positions 1227-1250 of COL4A4, containing 24 amino acid residues) is used as a repeating unit, and then 5-30 repeating units are tandemly linked to form recombinant type IV collagen; a vector containing nucleic acid molecules encoding recombinant type IV collagen is introduced into host cells, and efficient secretion-induced expression is carried out in host cell (especially commercial Pichia pastoris) expression systems to finally obtain a recombinant type IV collagen with high protein expression levels, which has good biological activities to promote cell migration and cell proliferation.

[0025] In this embodiment, the amino acid sequence of the repeating unit is shown in SEQ ID NO:1. Specifically, the amino acid sequence of the 24 amino acid residues, i.e., the repeating unit, is as follows: GKKGPPGPPGSSGPPGPAGATGRA.

[0026] Preferably, 5, 10, 15, 20, 25 or 30 repeating units are connected in series.

[0027] Specifically, host cells are cultured to express recombinant type IV collagen, and cultures containing recombinant type IV collagen are collected; the cultures containing recombinant type IV collagen are then separated and purified to obtain purified recombinant type IV collagen.

[0028] Specifically, the preparation methods for recombinant type IV collagen include: A nucleotide sequence encoding an amino acid sequence is inserted into an expression vector to obtain a recombinant plasmid vector. The recombinant plasmid vector is then introduced into host cells for culture to induce the expression of recombinant type IV collagen. After separation and purification, recombinant type IV collagen is obtained.

[0029] Preferably, the host cell can be a prokaryotic cell or a eukaryotic cell, including Escherichia coli, Bacillus subtilis or Bacillus licheniformis, Pichia pastoris, Saccharomyces cerevisiae, animal cells or plant cells; Escherichia coli and Pichia pastoris are preferred; Pichia pastoris is more preferred.

[0030] Preferably, the expression is one or both of constitutive expression and permeation expression. Specifically, the permeation inducer used in permeation expression is isopropyl-β-D-thiogalactoside (IPTG), β-galactoside, methanol, or ethanol.

[0031] Preferably, the separation and purification employs one or more of the following methods in combination: salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation. A combination of chromatographic chromatography and membrane separation, or a combination of ion exchange chromatography and membrane separation, is preferred.

[0032] The following examples further illustrate the recombinant type II collagen engineered bacteria that inhibit degradation according to the present invention.

[0033] Example 1 This embodiment provides a recombinant type IV collagen, which is formed by 10 repeating units connected in series.

[0034] In this embodiment, the amino acid sequence of the recombinant type IV collagen is shown in SEQ ID NO:2.

[0035] The amino acid sequence of recombinant type IV collagen (denoted as IV-2418) is as follows: GKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSGPPGPAGATGRA GKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSSGPPGPAGATGRAGKKGPPGPPGSGPPGPAGATGRA This embodiment provides a nucleic acid molecule encoding the above-mentioned recombinant type IV collagen, the nucleotide sequence of which is shown in SEQ ID NO:3 after optimization by Pichia pastoris codons.

[0036] The nucleotide sequence of recombinant type IV collagen (denoted as IV-2418) is as follows: This embodiment provides an expression vector containing the above-mentioned nucleic acid molecules.

[0037] Specifically, in this embodiment, the preparation method of recombinant type IV collagen includes the following steps: Step 1: The amino acid sequence shown in SEQ ID NO:2 was optimized according to the codons of the Pichia pastoris expression system to obtain the nucleotide gene sequence of recombinant collagen IV-2418 shown in SEQ ID NO:3. The obtained nucleotide gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized nucleotide gene sequence was ligated into the pPICZalphaA plasmid to obtain the pPICZalphaA-IV-2418 plasmid.

[0038] Step 2: Electroporate the pPICZalphaA-IV-2418 plasmid into Pichia pastoris X-33 competent cells. Use bleomycin as a selection marker to perform high copy screening and select the best transformant to obtain the Pichia pastoris expression strain.

[0039] Step 3: Select a single colony of the constructed Pichia pastoris expression strain and add it to 25 mL of BMMY liquid medium (1% yeast extract, 10% PBB, 1.34% YNB, 2% peptone and 1% glycerol). Incubate at 30℃ and 220 rpm for 24 h (OD of 10-20) for activation.

[0040] Transfer 1% of the inoculum to a 1000mL Erlenmeyer flask (containing 500mL of BMMY culture medium) and incubate at 30℃ and 220rpm for 24h (OD of 8-10) to serve as the seed for the next culture.

[0041] Prepare a high-density fermentation medium (3L) with the following composition: 120g glycerol, 27.3g K2SO4, 120g CaSO4 2H₂O 1.38g, KOH 6.3g, MgSO₄ 22.5g 7H2O, 40.2mL phosphoric acid, 2mL defoamer, 250g glycerin, and 250mL water.

[0042] Add 3L of culture medium to a 5L fermenter and sterilize at 121℃ for 20min. After cooling to 30℃, add 12mL of trace elements and adjust the pH to 5. Then, add the prepared upper tank seed to the fermenter by flame inoculation for fermentation culture.

[0043] When the OD value reaches 150-200, stop glycerol feeding. Measure the OD 30 min after stopping until the OD remains basically unchanged. Add methanol as an inducer and feed it with methanol periodically, adjusting the amount in real time according to the dissolved oxygen level. After induction for 40 h, end the fermentation. Centrifuge the culture medium at 12000 rpm for 2 min, collect the supernatant, and use the BCA method and SDS-PAGE method to detect protein yield and purity.

[0044] The collected fermentation supernatant was passed through a 10 kDa ultrafiltration membrane, then through an ion exchange column for further ultrafiltration to reduce conductivity, and finally freeze-dried to obtain recombinant type IV collagen.

[0045] In this embodiment, the protein content of recombinant type IV collagen (denoted as IV-10) was tested, and the results showed that the protein content of recombinant type IV collagen was 1.85 g / L. This demonstrates that the recombinant type IV collagen prepared in this embodiment was successfully secreted and expressed in the Pichia pastoris expression system.

[0046] Following the above preparation method, recombinant type IV collagen with 5 and 30 repetitions were constructed, respectively.

[0047] Furthermore, the biological function of the recombinant type IV collagen (denoted as IV-2418) obtained in Example 1 was evaluated through the following experiments.

[0048] Experiment 1: Structural Identification of Recombinant Type IV Collagen The recombinant type IV collagen pure product prepared in Example 1 was subjected to structural identification.

[0049] 1.1 SDS-PAGE electrophoresis Methods: The supernatant of the fermentation broth and the purified recombinant type IV collagen were subjected to SDS-PAGE electrophoresis (12% separating gel, 5% stacking gel), with a sample loading of 10 μg and electrophoresis at a constant voltage of 120 V. After electrophoresis, Coomassie Brilliant Blue R-250 staining was performed, and the band positions were observed after destaining. The target protein band in the SDS-PAGE gel was excised, digested in the gel, and identified by LC-MS / MS mass spectrometry.

[0050] The result is shown in the figure: Figure 1 The image shows the protein expressed in a 5 L collagen fermenter. The protein size is between 50 kDa and 70 kDa, indicating that the protein expressed after fermentation is the recombinant type IV collagen to be expressed in this embodiment. Further mass spectrometry identification... Figure 3 As shown, Figure 1 The protein at the displayed location has an amino acid sequence coverage of 87.5% with the recombinant type IV collagen shown in this example. Figure 2 The SDS-PAGE electrophoresis image shows a pure protein solution free of pigmented nucleic acids and other contaminants, produced after ion exchange in the separation and purification step. This image roughly confirms that the protein after ion exchange is still the desired target protein. Therefore, the above electrophoresis image demonstrates that the method of this embodiment can achieve the expression and preparation of recombinant type IV collagen.

[0051] The above SDS-PAGE electrophoresis and mass spectrometry results show that the molecular weight of the target protein band in the fermentation broth is consistent with the theoretical value, and the amino acid sequence coverage reaches 100%, confirming that the protein is the recombinant type IV collagen shown in SEQ ID NO.2 of this invention; the target protein with high purity can be obtained by one-step ion exchange chromatography.

[0052] 1.2 Ultraviolet Spectroscopy Analysis Methods: The purified recombinant type IV collagen was scanned across the entire wavelength range (200-600 nm) using a UV-Vis spectrophotometer.

[0053] The results are as follows Figure 4 As shown, the protein exhibits a characteristic absorption peak at approximately 202 nm, indicating that it possesses the typical ultraviolet absorption behavior of a protein polypeptide backbone.

[0054] 1.3 Circular Dichroism (CD) Spectroscopy Analysis Methods: Purified recombinant type IV collagen was dissolved in PBS buffer (pH 7.4) to prepare a solution with a concentration of 0.2 mg / mL. A circular dichroism spectroscopy was used to scan the wavelength range of 190–260 nm at 25 °C with a scan rate of 50 nm / min, a bandwidth of 1 nm, and a cuvette path length of 0.1 cm. PBS buffer was used as a blank control.

[0055] The results are as follows Figure 5 As shown, a distinct negative absorption peak appears at approximately 202 nm, indicating that the recombinant protein has the PPII helical conformation.

[0056] 1.4 Fourier Transform Infrared Spectroscopy (FTIR) Analysis Methods: Approximately 2 mg of recombinant type IV collagen lyophilized powder was compressed into transparent sheets. Fourier transform infrared spectroscopy was used for scanning, with a scanning range of 4000–400 cm⁻¹. -1 4 cm resolution -1 The number of scans was 32.

[0057] The results are as follows Figure 6 As shown, the IV-2418 protein is located at approximately 1645 cm⁻¹. -1A distinct absorption peak appears at approximately 1539 cm⁻¹, corresponding to the amide I band of the protein, mainly originating from the C=O stretching vibration in the peptide bond; at approximately 1539 cm⁻¹... -1 The characteristic absorption peak of amide II appears at approximately 1240 cm⁻¹, mainly related to the bending vibration of NH₄⁺ and the stretching vibration of CN₂; simultaneously, at approximately 1240 cm⁻¹... -1 An absorption peak of amide III was observed at this region, which is associated with CN stretching vibration and NH bending vibration in the collagen structure.

[0058] The three characteristic absorption peaks mentioned above are typical infrared spectral features of collagen, further confirming that this recombinant type IV collagen has the secondary structure characteristic of collagen.

[0059] The circular dichroism spectroscopy and Fourier transform infrared spectroscopy analyses described above have confirmed that the recombinant type IV collagen prepared in this invention has typical secondary structure characteristics of collagen.

[0060] To further verify the biological function of this protein, its activity in promoting cell proliferation was measured.

[0061] Experiment 2: Assay of the cell proliferation activity of recombinant type IV collagen Experimental method: L929 cells that have grown to 70%-80% of the bottom area of ​​the culture flask were taken, digested with 0.25% trypsin containing EDTA, and then cultured in complete medium to prepare a cell density of 8×10⁻⁶ cells / year. 4 Cell suspension at 100 μL / mL. Seed 100 μL of cell suspension into 96-well culture plates and incubate at 37°C with 5% CO2 and saturated humidity. After 24 h of cell culture, aspirate the complete culture medium.

[0062] Different concentrations of recombinant humanized type IV collagen solution (0.1, 0.5, 1, 3, 5 mg / mL, with 5 replicates for each concentration) diluted in DMEM / F-12 medium were added to the experimental group. The control group consisted of cells cultured in DMEM / F-12 medium, and the blank group consisted of cells-free DMEM / F-12 medium. The cells were cultured for 24 h in an incubator at 37°C and 5% CO2 saturated humidity.

[0063] After aspirating the culture medium from each group, add 110 μL of a mixture of DMEM / F-12 medium and L929 (100 μL medium, 10 μL L929), and incubate in a cell culture incubator for 1–4 h. Measure the absorbance (OD value) of each well at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate cell viability based on the mean absorbance of each group using the following formula: RGR (%) = Experimental group (Blank group) Control group (Blank group) × 100% The results are as follows Figure 7 As shown, compared with the control group (100%), the relative cell proliferation rate of the recombinant type IV collagen experimental group prepared in this invention was greater than 100% at low concentrations, and showed a trend of first promoting and then inhibiting proliferation with increasing concentration. This indicates that the recombinant type IV collagen prepared in this invention can promote the proliferation of L929 cells.

[0064] Experiment 3: Assay of the cell migration activity of recombinant type IV collagen Experimental method: L929 cells that had grown to 70%-80% of the culture dish area were digested with 0.25% trypsin and then cultured in complete medium to achieve a cell density of 2.5 × 10⁻⁶ cells / mL. 5 Cell suspension of cells / mL. Draw three horizontal lines on the bottom of the well plate using a marker and a ruler. Seed 2 mL of cell suspension into a 6-well plate and incubate at 37°C with 5% CO2 saturated humidity.

[0065] After 24 h of cell culture, using a 10 μL pipette tip aligned vertically with a ruler, gently push downwards to create longitudinal scratches in the wells. Aspirate the complete culture medium, wash three times with PBS, and remove the scratched cells. In the experimental group, a 3 mg / mL solution of recombinant type IV collagen prepared in Example 1, diluted in serum-free DMEM / F-12 medium (with three replicates), was added. The control group consisted of cells in serum-free DMEM / F-12 medium without added recombinant type IV collagen.

[0066] The samples were incubated at 37℃ and 5% CO2 saturated humidity for 0 h and 24 h, respectively. The intersection of the horizontal and vertical lines was used as the core, and the images were taken under a 10x microscope to obtain images of 9 parts, with a total of 27 data points for each combination.

[0067] The results are as follows Figure 8 As shown, the central scratch area was cell-free at 0 h, but after culturing in recombinant type IV collagen solution for 24 h, L929 cells migrated to the central scratch area. The results indicate that the recombinant type IV collagen prepared in Example 1 has the ability to promote L929 cell migration.

[0068] Based on the above experimental results, the recombinant type IV collagen expressed in this invention has the ability to promote the migration and proliferation of mouse fibroblasts, thereby achieving wound repair and cartilage healing. In addition, the recombinant type IV collagen also contains natural amino acid sequences with multiple functional sites, uniform molecular weight, good solubility, and good cell adhesion and proliferation properties, and can be applied in a variety of fields.

[0069] The recombinant type IV collagen expressed in this invention can be used in the preparation of skin care products, health products, medical devices, and tissue engineering materials. Specifically, skin care products include cosmetics or invasive beauty products; medical devices include medical dressings, medical spray dressings, medical gel dressings, or medical lyophilized powder dressings; and tissue engineering materials include subcutaneous fillers, artificial bone and artificial skin, oral absorbable biomembranes, bone implants, vascular scaffolds, hemostatic agents, procoagulants, intercellular matrix scaffolds, or collagen sponges.

[0070] The above are several preferred embodiments of the preparation method of the present invention, but they should not be regarded as limitations on the technical solutions protected by the present invention. Any alternative solutions obtained by those skilled in the art based on the technical ideas of the present invention without creative labor should fall within the protection scope of the present invention.

Claims

1. A recombinant type IV collagen with cell migration and proliferation promoting activities, characterized in that, The recombinant type IV collagen is formed by multiple repeating units connected in series, and the amino acid sequence of the repeating units is shown in SEQ ID NO:

1.

2. The recombinant type IV collagen according to claim 1, characterized in that, The number of repeating units is 5-30; preferably, the number of repeating units is 10.

3. The recombinant type IV collagen according to claim 2, characterized in that, The amino acid sequence of the recombinant type IV collagen is shown in SEQ ID NO.

2.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant type IV collagen as described in any one of claims 1-3.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4.

6. A host cell, characterized in that, The host cell comprises the recombinant type IV collagen as described in any one of claims 1-3, the nucleic acid molecule as described in claim 4, or the expression vector as described in claim 5; the host cell is a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is Escherichia coli; the eukaryotic cell is Pichia pastoris.

7. A method for preparing recombinant type IV collagen with cell migration and proliferation-promoting activities, characterized in that, Includes the following steps: Step 1: Insert the nucleotide sequence encoding the amino acid sequence of claim 3 into the vector to obtain the expression vector; Step 2: Introduce the expression vector into the host cells described in claim 6 and culture them; Step 3: Induce expression of recombinant type IV collagen, then separate and purify it to obtain the recombinant type IV collagen.

8. The method for preparing recombinant type IV collagen according to claim 7, characterized in that, The inducing agent used in step 3 is any one of isopropyl-β-D-thiogalactoside, β-galactoside, methanol, or ethanol; more preferably, the inducing agent is methanol; the separation and purification in step 3 is one or a combination of salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, and membrane separation; preferably, the chromatographic chromatography is ion exchange chromatography.

9. The use of the recombinant type IV collagen according to any one of claims 1-3 in the preparation of cosmetics, medical devices or tissue engineering materials that promote cell migration and proliferation.

10. The application according to claim 9, characterized in that, The medical devices include medical dressings, medical spray dressings, medical gel dressings, or medical lyophilized powder dressings; the tissue engineering materials include cell culture substrates, wound repair materials, or tissue engineering scaffold materials.

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