Soluble recombinant triple helix III type collagen, preparation method and application

Abstract

The invention belongs to the technical field of gene engineering, and discloses soluble recombinant triple helix III type collagen, a preparation method and application, the amino acid sequence of the recombinant triple helix III type collagen is shown as SEQ ID No.1, compared with animal source III type collagen, the recombinant triple helix III type collagen is single in component, high in purity, small in batch-to-batch difference, free of immunogenicity, free of virus transmission and other hidden dangers; the constructed escherichia coli of the Sumo-III type collagen is short in culture period, the expression quantity of the III type collagen is high, and inclusion bodies are not easy to form; the water solubility is good, and the quality is stable; a Gly-X-Y repeated amino acid sequence mode is strictly obeyed, and the disease risk caused by mutation does not exist; and the three-helix structure can be widely applied to the fields of skin care products, skin repair dressings, implants, artificial skin, biological materials, medical instruments, health food and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a soluble recombinant triple-helix type III collagen, a preparation method and an application thereof. Background Art

[0002] Collagen is the most abundant protein in humans and animals, accounting for about 25-30% of the total protein. At present, 28 types of collagen have been found in vertebrates and invertebrates. Among them, type III collagen is mainly distributed in tissues such as skin, blood vessels, tendons, and muscles, and it plays a key role in maintaining the elasticity and tension of these tissues. Compared with other types of collagen, type III collagen usually plays a more important role in tissue repair, wound healing, protecting blood vessels and nerves, improving cell microenvironment, and promoting tissue regeneration. Biomaterials with type III collagen as the main raw material are widely used in fields such as tissue engineering and regenerative medicine.

[0003] At present, collagen is mainly extracted from animal tissues, which has many hidden dangers such as immunogenicity, large batch-to-batch differences, virus and disease transmission, and allergic reactions. Compared with type I collagen, the content of type III collagen in tissues is relatively low, and the extraction of type III collagen from animal tissues is difficult, costly, and low in purity. In addition, type III collagen is a homotrimeric protein composed of three identical α(III) chains. This triple helix structure gives type III collagen a certain degree of flexibility and elasticity. Traditional collagen extraction methods mainly use different treatment methods such as acid, alkali or protease to treat animal tissues to obtain collagen. These methods are easy to destroy the triple helix structure and biological activity of type III collagen.

[0004] Recombinant collagen prepared by genetic engineering technology can replace collagen extracted from heterologous species and be applied in the field of medical regeneration because of its fixed preparation process, single composition and small batch-to-batch differences. At present, a variety of expression systems such as transgenic animals, plants, mammalian cells, bacteria, and yeast can be used for the preparation of recombinant collagen. Compared with other systems, the E. coli expression system has the advantages of short culture cycle, low cost, and high expression level, and is the first choice for industrial production of collagen. For example, Chinese patent CN110903383A discloses a recombinant human type I collagen, a coding gene, an engineered bacterium and its application in the preparation of collagen preparations, wherein the amino acid sequence is a peptide segment GSKGDTGEPGPVGVQGPPGPAGEEGKRGARGEP repeated 9 times; Chinese patent CN105061589A provides a recombinant human type I collagen, including a peptide segment 660-964 of the α1 chain of human type I collagen, 15 amino acids at positions 680-694 at the N-terminus, and 15 amino acids at positions 950-964 at the C-terminus and 3 groups of GER amino acid triplets. However, the recombinant type I collagen prepared by these methods lacks the characteristic triple helical structure and biological function of collagen.

[0005] More importantly, type III collagen has a complex triple-helix structure, which usually cannot be correctly folded in the Escherichia coli prokaryotic expression system, causing it to lose its biological activity during the expression process, forming insoluble aggregates and eventually inclusion bodies. In addition, the characteristics of collagen itself will also cause it to easily aggregate into particles in the cell when expressed in Escherichia coli, and eventually form inclusion bodies, which is not conducive to the subsequent purification of collagen.

[0006] In view of the above technical problems, the present invention provides a soluble recombinant triple-helical type III collagen, a preparation method and an application thereof. The soluble recombinant triple-helical type III collagen has a typical triple-helical structure of collagen and strictly follows the Gly-XY repeating sequence. The soluble recombinant triple-helical type III collagen constructed by the present invention is easy to purify in an Escherichia coli expression system, and the prepared product has good water solubility and stable quality, and can be widely used in skin care products, skin repair dressings, implants, artificial skin, biomaterials, medical devices, health foods and other fields. Summary of the invention

[0007] In view of the above technical problems, the present invention aims to provide a soluble recombinant triple-helical type III collagen, the amino acid sequence of which is shown in SEQ ID No.1.

[0008] Preferably, the nucleotide sequence of the recombinant triple-helical type III collagen is as shown in SEQ ID No.2.

[0009] The second object of the present invention is to provide a recombinant vector or a recombinant genetically engineered bacterium carrying the nucleotide sequence of the recombinant triple-helical type III collagen.

[0010] Preferably, the vector includes pCold or pET, and the engineered bacteria is Escherichia coli.

[0011] The third object of the present invention is to provide a method for preparing the triple helical type III collagen, wherein the type III collagen is obtained by treating a precursor collagen with a protease, and the precursor collagen is precursor collagen Sumo-III and / or precursor collagen MBP-III.

[0012] Preferably, the amino acid sequence of the precursor collagen Sumo-III is shown as SEQ ID No.3, and the nucleotide sequence is shown as SEQ ID No.4; the amino acid sequence of the precursor collagen MBP-III is shown as SEQ ID No.5, and the nucleotide sequence is shown as SEQ ID No.6.

[0013] Preferably, the protease includes one or more of pepsin, trypsin, papain, bromelain and thrombin.

[0014] Preferably, the method is specifically:

[0015] (1) Synthesizing gene sequences encoding precursor collagen Sumo-III and precursor collagen MBP-III, wherein the nucleotide sequence of the precursor collagen Sumo-III is shown in SEQ ID No. 4; and the nucleotide sequence of the precursor collagen MBP-III is shown in SEQ ID No. 6;

[0016] (2) connecting the gene sequence and the vector described in step (1), transforming Escherichia coli, and obtaining a recombinant genetically engineered bacterium;

[0017] (3) culturing the recombinant genetically engineered bacteria constructed in step (2), inducing them to express collagen, collecting bacterial precipitates, crushing them to obtain supernatant, and purifying them through a nickel column to obtain precursor collagen Sumo-III and precursor collagen MBP-III;

[0018] (4) adding protease to treat the precursor collagen Sumo-III and precursor collagen MBP-III obtained in step (3), removing the N-terminal solubility-promoting tag and the purification tag, and obtaining type III collagen;

[0019] (5) The type III collagen obtained in step (4) is purified to obtain high-purity soluble recombinant triple-helical type III collagen.

[0020] Preferably, the amino acid sequences of the Sumo-III and MBP-III precursor collagens are shown in SEQ ID NO.3 and SEQ ID NO.5.

[0021] Preferably, the purification in step (3) is performed by affinity chromatography.

[0022] Preferably, the purification in step (5) is performed by DEAE-52 anion chromatography.

[0023] The fourth object of the present invention is to provide the use of the recombinant triple-helical type III collagen in the preparation of skin repair dressings, implants, biomaterials, and medical devices.

[0024] The fifth object of the present invention is to provide the use of the recombinant triple-helical type III collagen in the preparation of cosmetics, health products and foods.

[0025] The sixth object of the present invention is to provide the use of the recombinant triple-helix type III collagen in the preparation of artificial skin, artificial bone, artificial cartilage and artificial cornea.

[0026] The beneficial effects of the present invention are as follows: (1) The present invention provides a soluble recombinant triple-helical type III collagen, which, compared with animal-derived type III collagen, has a single component, high purity, small batch-to-batch differences, no immunogenicity, no hidden dangers such as virus transmission; (2) The Escherichia coli strain of the soluble recombinant triple-helical Sumo-III type collagen constructed by the present invention has a short culture cycle, a high expression level of type III collagen, and is not easy to form inclusion bodies; (3) The soluble recombinant triple-helical type III collagen provided by the present invention has good water solubility, stable quality, and pepsin resistance; (4) The soluble recombinant triple-helical type III collagen strictly complies with the Gly-XY repeating amino acid sequence pattern, and there is no risk of disease caused by mutation; (5) The soluble recombinant triple-helical type III collagen has a characteristic triple-helical structure of collagen; (6) The soluble recombinant triple-helical type III collagen can promote cell proliferation and migration, and has low cytotoxicity, and can be widely used in skin care products, skin repair dressings, implants, artificial skin, biomaterials, medical devices, health foods and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments with reference to the following drawings.

[0028] Figure 1SDS-PAGE identification of III, Sumo-III and MBP-III collagen expressed in Escherichia coli. Legend: From left to right, lane 1: marker; lanes 2, 3, and 4 are the supernatant, precipitate, and type III collagen purified from the supernatant after the recombinant type III collagen was expressed in Escherichia coli after being crushed; lanes 5, 6, and 7 are the supernatant, precipitate, and Sumo-III precursor collagen purified from the supernatant after the recombinant type III collagen was expressed in Escherichia coli after being crushed; lanes 8, 9, and 10 are the supernatant, precipitate, and MBP-III precursor collagen purified from the supernatant after the recombinant type III collagen was expressed in Escherichia coli after being crushed.

[0029] Figure 2 Purification and SDS-PAGE identification of recombinant triple-helical Sumo-III precursor collagen. Figure legends: 1: supernatant after disruption of recombinant Sumo-III type collagen Escherichia coli expression cells; 2: flow-through after the supernatant after disruption of recombinant Sumo-III type collagen Escherichia coli expression cells was bound to the nickel column; 3: flow-through after the nickel column was rinsed for the second time with PBS buffer containing 20 mM imidazole; 4: flow-through after the nickel column was rinsed for the fourth time with PBS buffer containing 100 mM imidazole; 5: Sumo-III precursor collagen solution collected after elution of the nickel column with PBS buffer containing 500 mM imidazole. Figure 3 SDS-PAGE identification of recombinant triple-helical Sumo-III precursor collagen by protease digestion. Legend: 1: sample of 2 mg / mL Sumo-III precursor collagen solution; 2: sample after 2 mg / mL Sumo-III precursor collagen solution was treated with 0.01 mg / mL pepsin at 4℃ for 1 hour; 3: sample after 2 mg / mL Sumo-III precursor collagen solution was treated with 0.01 mg / mL pepsin at 4℃ for 2 hours; 4: sample after 2 mg / mL Sumo-III precursor collagen solution was treated with 0.01 mg / mL pepsin at 4℃ for 4 hours; 5: sample after 2 mg / mL Sumo-III precursor collagen solution was treated with 0.01 mg / mL pepsin at 4℃ for 5 hours; 6: sample after 2 mg / mL Sumo-III precursor collagen solution was treated with 0.01 mg / mL pepsin at 4℃ for 17 hours.

[0030] Figure 4SDS-PAGE identification of recombinant triple-helical type III collagen after anion exchange purification. Legend: from left to right, lane 1: marker; lane 2: Sumo-III collagen solution after 17h of enzymatic digestion with 0.01mg / mL pepsin; lane 3: flow-through after binding of the type III collagen solution obtained after enzymatic digestion with a DEAE-52 column; lane 4: the eluate after eluting the DEAE-52 column bound to type III collagen with a NaH2PO4-Na2HPO4 buffer at a pH of 6.5; lanes 5, 6, 7, 8, 9, and 10: the eluate after eluting the DEAE-52 column bound to type III collagen with a NaH2PO4-Na2HPO4 buffer at a pH of 6.5 containing 0.1M, 0.2M, 0.3M, 0.5M, 0.7M, and 1M NaCl, respectively.

[0031] Figure 5 SDS-PAGE identification of recombinant triple helical Sumo-III procollagen protein by protease digestion. Legend: from left to right, the first lane: marker; the second lane: type III collagen purified by ion exchange;

[0032] Lanes 3, 4, 5, 6, 7, and 8: samples of type III collagen purified by enzymatic digestion and ion exchange treatment with 0.01 mg / mL pepsin at 4°C for 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h;

[0033] Figure 6 Native-PAGE of recombinant triple-helical type III collagen. Legend: From left to right, lane 1: marker; lane 2: Native-PAGE identification of type III collagen obtained after enzymatic digestion of Sumo-III precursor collagen;

[0034] Figure 7 Schematic diagram of the isoelectric point of recombinant triple helical type III collagen

[0035] Note: The sample is the type III collagen solution obtained after enzymatic digestion of Sumo-III precursor collagen;

[0036] Figure 8 Circular dichroism spectra of recombinant triple helical type III collagen

[0037] Note: The sample is the type III collagen solution obtained after enzymatic digestion of Sumo-III precursor collagen;

[0038] Fig. 9 Cytotoxicity of recombinant triple-helical type III collagen

[0039] Fig.10Cell proliferation diagram of recombinant triple helical type III collagen

[0040] Fig.11 a is the cell migration diagram of recombinant triple helical type III collagen, and b is the statistical diagram of cell migration rate of type III collagen. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be described in detail in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0042] Unless otherwise specified, the experimental materials in the following examples can be purchased on the market.

[0043] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0044] Example 1: Fermentation of soluble recombinant triple helical type III collagen strain

[0045] The amino acid sequence of the soluble recombinant triple-helical type III collagen described in the present invention is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.2. It can be obtained by treating precursor collagens Sumo-III and MBP-III with proteases. The amino acid sequences of precursor collagens Sumo-III and MBP-III are shown in SEQ ID NO.3 and SEQ ID NO.5, respectively, and their gene sequences are shown in SEQ ID NO.4 and SEQ ID NO.6, respectively.

[0046] 1. The amino acid sequences of the two precursor collagens were determined as SEQ ID NO.3 and SEQ ID NO.5, respectively. After optimizing the codon preference of Escherichia coli, their gene sequences were determined as SEQ ID NO.4 and SEQ ID NO.6, respectively.

[0047] 2. Construction of E. coli expression strain for procollagen expression:

[0048] The gene sequences of the synthesized precursor collagen, as shown in SEQ ID NO.4 and SEQ ID NO.6, were constructed into the E. coli expression vector pCold, and the plasmid was successfully synthesized by DNA sequencing; the plasmid was transformed into the E. coli BL21-DE3 strain to screen and obtain the precursor collagen expression strain, and the successfully transformed strain was stored in glycerol at -80°C;

[0049] 3. Fermentation of Precursor Collagen:

[0050] The cryopreserved Sumo-III procollagen expression strain was inoculated into 50 ml LB medium (containing 50 μg / mL kanamycin) at a ratio of 1:1000 and cultured overnight at 37°C and 200 rpm. Subsequently, when the bacteria were in the middle growth phase, the bacteria were inoculated into 1 L LB medium (containing 50 μg / mL kanamycin) at a ratio of 1:50 and continued to be cultured at 37°C and 200 rpm until the optical density (OD) of the bacterial culture reached 0.5-2.0. At this time, 1 mM / mL IPTG (isopropyl β-D-thiogalactoside) was added for induction, and the culture was transferred to 25°C and 200 rpm for 16 hours, and the bacteria were collected by centrifugation.

[0051] Example 2: Purification of soluble recombinant triple helical type III collagen strain

[0052] 1. Bacterial cell disruption: The collected bacteria were dispersed with PBS buffer (PH 7.4) containing 0.02 M imidazole at a dilution ratio of 1:10, and the bacteria were disrupted three times under 800 bar conditions, and the supernatant was collected by centrifugation.

[0053] 2. Purification of procollagen by nickel column affinity chromatography: The supernatant obtained in step 1 above was bound to a nickel column, and the impurities were eluted with PBS buffer (PH 7.4) containing 0.05 M imidazole, and the procollagen was eluted with PBS buffer (PH 7.4) containing 0.5 M imidazole.

[0054] 3. The precursor collagen obtained by nickel column purification was diluted to a certain concentration, pepsin was added, and the mixture was reacted at 4°C for more than 12 hours to remove the N-terminal tag of the precursor collagen.

[0055] 4. DEAE-52 anion purification to remove the cleavage product: The cleavage product obtained in the above step 3 is dialyzed overnight in 0.02MNaH2PO4-Na2HPO4 buffer (PH 6.5), and then combined with a DEAE-52 anion exchange column. The impurities are eluted with a NaH2PO4-Na2HPO4 buffer solution containing 0.01M NaCl for 15 min / time, and the impurities are washed twice; the type III collagen is eluted and collected with a NaH2PO4-Na2HPO4 buffer solution containing 0.05MNaCl for 30 min / time, and the type III collagen is collected twice; high-purity soluble recombinant triple-helical type III collagen can be obtained.

[0056] Example 3: Characterization of soluble recombinant triple-helical type III collagen

[0057] 1. SDS-PAGE Characterization of Soluble Recombinant Triple-helical Type III Collagen

[0058] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to characterize the expression of the Sumo-III procollagen protein strain. The expression level of Sumo-III procollagen protein in the supernatant after the disruption was the highest, which significantly increased the soluble expression of type III collagen (such as Figure 1 shown).

[0059] The precursor Sumo-III collagen was characterized by SDS-PAGE after nickel column purification. Figure 2 As shown, Sumo-III procollagen can be recovered in the eluate after nickel column purification.

[0060] The III collagen obtained by digesting Sumo-III precursor collagen with pepsin was characterized. Figure 3 As shown, the N-terminal tag of Sumo-III procollagen was removed by pepsin treatment, and the size of the protein after enzyme cleavage was about 27 kDa.

[0061] The III collagen was characterized after purification by DEAE-52 anion exchange. Figure 4 As shown, after two purifications, the type III collagen was a single band with an apparent molecular weight of 28 KDa, indicating that high-purity recombinant humanized type III collagen was successfully prepared.

[0062] The purified soluble recombinant triple helical type III collagen was characterized by SDS-PAGE. The results showed that the soluble recombinant triple helical type III collagen was still a single band after being treated with 0.01 mg / mL pepsin at 4°C for 24 hours, indicating that it has protease resistance and stable quality. Figure 5 shown.

[0063] 2. Native-PAGE Characterization of Soluble Recombinant Triple-helical Type III Collagen

[0064] Native-PAGE was used to characterize soluble recombinant triple-helical type III collagen. Native-PAGE requires that the natural integrity of the sample be maintained as much as possible during the experiment. Therefore, the type III collagen that was lyophilized after enzyme digestion was mixed with a loading buffer that did not contain SDS and mercaptoethanol. No heating was required. The buffer maintained the secondary structure and natural charge density of the protein. SDS was removed from the concentrated gel, separation gel, and electrophoresis buffer, and the concentration of the separation gel was 10%. The sample mixture was loaded into the gel and the voltage was set to 70V for electrophoresis. The electrophoresis system was placed on ice and a higher voltage was not set to prevent protein degradation. After the electrophoresis, the sample was stained with Coomassie Brilliant Blue dye and photographed to save the results.

[0065] Native-PAGE results showed that the soluble recombinant triple-helical type III collagen migrated to a position of about 100KDa in the native polyacrylamide gel, which is about three times the apparent molecular weight of its single-chain molecule, indicating that the soluble recombinant triple-helical type III collagen we obtained is a trimer (such as Figure 6 shown).

[0066] 3. Zeta Characterization of Soluble Recombinant Triple-helical Type III Collagen

[0067] The isoelectric point of a protein affects its charge state in solutions of different pH values ​​and has an important influence on its solubility, stability, and other aspects. In addition, determining the isoelectric point of a protein can be used to optimize the protein purification process. Near the isoelectric point, the protein presents a net charge of zero, at which point the interaction between the protein and the ions in the solution is minimal, and the protein concentration in the solution is the lowest. Therefore, by adjusting the pH value of the solution, the protein can be precipitated or aggregated near the isoelectric point, thereby facilitating its purification.

[0068] The soluble recombinant triple helical type III collagen after nickel column purification and enzyme digestion was prepared into a solution with a concentration of 0.5 mg / mL, divided into 18 portions, and the pH thereof was adjusted to 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10, respectively. The isoelectric point was measured using a 90Plus Pals Zeta potential and particle size analyzer. The isoelectric point was measured to be 5.2 (eg Figure 7 Under these conditions, a variety of ion exchange media can be selected for purification.

[0069] 4. Circular dichroism characterization of soluble recombinant triple-helical type III collagen

[0070] Circular dichroism is a commonly used method for characterizing protein structure. Soluble recombinant triple helical type III collagen was prepared into an aqueous solution with a concentration of 1 mg / mL. The circular dichroism spectrum was scanned at full wavelength (190-260 nm) at 4°C using a cuvette with a slit of 1 mm. The wavelength interval was 1 nm and the dwell time was 5 s at each wavelength. The results showed that the soluble recombinant triple helical type III collagen prepared by the present invention had a positive peak at 225 nm and a negative peak at 192 nm, which was consistent with the typical collagen circular dichroism spectrum, indicating that the soluble recombinant triple helical type III collagen had a triple helical structure (such as Figure 8 shown).

[0071] Example 4. Biological activity of soluble recombinant triple-helical type III collagen

[0072] 1. Cytotoxicity Assay

[0073] 100 μL of the solution was diluted to a density of 1×10 5 HDF-1 cells were added to a 96-well plate and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The recombinant triple helix type III collagen purified in Example 2 was prepared into protein solutions with concentrations of 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, and 0.5 mg / mL using DMEM high-glucose medium, and sterilized by filtration with a 0.22 μm sterile filter. The culture medium in the 96-well plate was aspirated, and 100 μL of the prepared recombinant triple helix type III collagen solution of different concentrations was added to the 96-well plate, and duplicate wells were set. Only DMEM high-glucose medium was added to the control group. Continue to culture in an incubator at 37°C and 5% CO2 for 24 hours, and use CCK-8 to detect whether the recombinant triple helix type III collagen is toxic to human skin fibroblasts.

[0074] like Fig. 9 As shown, different concentrations of recombinant triple helical type III collagen solutions can promote the proliferation of HDF-1 cells, but the ability of promoting the proliferation of HDF-1 cells is stronger when the concentration of the recombinant triple helical type III collagen solution is 0.25 mg / mL.

[0075] 2. Cell Proliferation Assay

[0076] 100 μL of the solution was diluted to a density of 1×10 5HDF-1 cells were added to a 96-well plate and cultured in an incubator at 37°C and 5% CO2 for 24 hours, and the culture medium in the 96-well plate was aspirated. The recombinant triple helical type III collagen purified in Example 2 and the bovine type III collagen extracted from tissue COL III were prepared into a protein solution with a concentration of 0.25 mg / mL using DMEM high-glucose culture medium, and filtered and sterilized with a 0.22 μm sterile filter, added to a 96-well plate, and duplicate wells were set. Only DMEM high-glucose culture medium was added to the control group. Continue to culture in an incubator at 37°C and 5% CO2 for 24 hours, and use CCK-8 to detect the proliferation-promoting effect of recombinant triple helical type III collagen on human skin fibroblasts HDF-1.

[0077] like Fig.10 As shown, the recombinant triple helical type III collagen III has a significant effect of promoting the proliferation of human skin fibroblasts HDF-1, and is superior to tissue-extracted type III collagen COl III.

[0078] 3. Cell Migration Assay

[0079] The cell scratch method was used to determine the cell migration promoting effect of the recombinant triple helical type III collagen prepared in Example 2. 2 mL of a 2.5×10 5 HDF-1 cells were added to a six-well plate and cultured for 24 hours until the cell confluence rate reached 95%-100%. Use a 10μL gun tip to align with a ruler and gently push vertically on the bottom of the 6-well plate to form a scratch. Aspirate the culture medium. The recombinant triple helical type III collagen purified in Example 2 and the bovine type III collagen extracted from tissue COL III were prepared into a protein solution with a concentration of 0.25 mg / mL using DMEM high-glucose culture medium, and 2 mL was added to two wells of the 6-well plate respectively. And 2 mL of DMEM high-glucose culture medium was added to one well of the 6-well plate as a control. The changes in the scratch area at 0h and 24h were recorded under an inverted microscope.

[0080] like Fig.11 As shown, compared with the blank control group, the recombinant triple helical type III collagen III exhibited a good cell migration promoting effect.

[0081] In summary, the present invention provides a soluble recombinant triple-helical type III collagen, which, compared with animal-derived type III collagen, has a single component, high purity, small batch-to-batch differences, no immunogenicity, no hidden dangers such as virus transmission; the Escherichia coli strain of the soluble recombinant triple-helical Sumo-III type collagen constructed by the present invention has a short culture cycle, a high expression level of type III collagen, and is not easy to form inclusion bodies; the soluble recombinant triple-helical type III collagen provided by the present invention has good water solubility, stable quality, and pepsin resistance; the soluble recombinant triple-helical type III collagen strictly adheres to the Gly-XY repeating amino acid sequence pattern, and there is no disease risk caused by mutation; the soluble recombinant triple-helical type III collagen has a characteristic triple-helical structure of collagen; the soluble recombinant triple-helical type III collagen can promote cell proliferation and migration, and has low cytotoxicity, and can be widely used in skin care products, skin repair dressings, implants, artificial skin, biomaterials, medical devices, health foods and other fields.

Claims

1. A soluble recombinant triple-helical type III collagen, characterized in that: The amino acid sequence of the recombinant triple-helical type III collagen is shown in SEQ ID No.

1.

2. The recombinant triple helical type III collagen according to claim 1, characterized in that The nucleotide sequence of the recombinant triple-helical type III collagen is shown in SEQ ID No.

2.

3. A recombinant vector or recombinant genetically engineered bacterium carrying the nucleotide sequence of the recombinant triple-helical type III collagen as claimed in claim 2.

4. The recombinant vector or recombinant genetically engineered bacterium according to claim 3, characterized in that: The vector includes pCold or pET, and the engineering bacteria is Escherichia coli.

5. The method for preparing triple helical type III collagen according to claim 1, characterized in that: The type III collagen is obtained by treating a precursor collagen with a protease. The precursor collagen is precursor collagen Sumo-III and / or precursor collagen MBP-III. The amino acid sequence of the precursor collagen Sumo-III is shown in SEQ ID No.3, and the amino acid sequence of the precursor collagen MBP-III is shown in SEQ ID No.

5.

6. The preparation method according to claim 5, characterized in that: The nucleotide sequence of the precursor collagen Sumo-III is shown in SEQ ID No.4; the nucleotide sequence of the precursor collagen MBP-III is shown in SEQ ID No.

6.

7. The preparation method according to claim 5, characterized in that: The protease includes one or more of pepsin, trypsin, papain, bromelain and thrombin.

8. Use of the recombinant triple helical type III collagen as claimed in claim 1 in the preparation of skin repair dressings, implants, biomaterials and medical devices.

9. Use of the recombinant triple helical type III collagen as claimed in claim 1 in the preparation of cosmetics, health products and foods.

10. Use of the recombinant triple helical type III collagen as claimed in claim 1 in the preparation of artificial skin, artificial bone, artificial cartilage and artificial cornea.

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