Recombinant collagen with enhanced skin regeneration ability, preparation method and application thereof

Abstract

The present invention discloses a recombinant collagen with enhanced skin regeneration ability, a preparation method thereof and an application thereof. The present invention provides a recombinant type III collagen based on the gene sequence of human type III collagen α1 chain, and the encoding nucleic acid molecule sequence thereof is shown in SEQ NO.1. The recombinant type III collagen is expressed by high-density fermentation of Pichia pastoris GS115 strain, combined with optimized cation exchange chromatography and anion exchange chromatography purification processes, to achieve efficient preparation with a protein purity of 99.2% and a yield of 13.8 g / L; the recombinant type III collagen is highly consistent with natural collagen, and can significantly promote skin regeneration, collagen fiber deposition and wound repair.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and specifically to a recombinant collagen protein having the ability to enhance skin regeneration, a preparation method thereof and an application thereof. Background Art

[0002] Collagen is one of the main structural proteins in the human body. It is widely distributed in the skin, bones, muscles and connective tissues, and plays a role in supporting and restoring elasticity. Collagen is an important component of the skin matrix. It can provide support and structural basis for skin cells, promote cell proliferation and migration, and significantly help skin regeneration, especially in wound repair, skin elasticity restoration and anti-aging. Therefore, collagen has become an important research object in medical fields such as skin regeneration and care.

[0003] Traditional collagen is mainly derived from animal tissues, but collagen from this source has problems such as immune rejection and potential animal virus risks. For this reason, scientists began to study recombinant collagen as an alternative. Recombinant collagen has the advantages of low immune rejection, no potential animal virus risks, good water solubility, and large-scale production, and has gradually become a research hotspot.

[0004] In recent years, the production of recombinant type III collagen using genetic engineering technology has become an important research direction. The supplementation of type III collagen can accelerate the healing of skin wounds and promote the formation of new skin. In addition, it can also promote the repair of extracellular matrix and enhance the stability of the skin. Through gene synthesis technology, the gene sequence of human type III collagen α1 chain is cloned into an appropriate expression vector and expressed using prokaryotic or eukaryotic cell systems. These expression systems include Escherichia coli, mammalian cells, insect cells, plant cells and yeast expression systems. In contrast, the yeast expression system has become an ideal choice for the production of recombinant type III collagen due to its advantages such as efficient protein expression ability, easy genetic modification, low cost and high cell density fermentation.

[0005] However, in the prior art, the research on how to select gene sequences, expression vectors, fermentation and purification processes to obtain recombinant type III collagen with significantly enhanced skin regeneration function is relatively one-sided and difficult to serve as a guide. In addition, whether the prior art can achieve high protein yield and high purity preparation of recombinant type III collagen is not involved.

[0006] Therefore, developing a recombinant type III collagen with enhanced skin regeneration ability is an issue that needs to be urgently addressed in the industry. Summary of the invention

[0007] The technical problem to be solved by the present invention is to target the recombinant type III collagen protein lacking the ability to enhance skin regeneration. To this end, the present invention provides a method for preparing recombinant type III collagen protein with high protein yield and high purity, wherein the type III collagen protein has the ability to enhance skin regeneration.

[0008] The present invention solves the above technical problems through the following technical solutions.

[0009] The present invention provides a recombinant type III collagen protein having the ability to enhance skin regeneration. The amino acid sequence of the recombinant type III collagen protein is shown in SEQ NO.1.

[0010] The present invention also provides a nucleic acid molecule encoding the above-mentioned recombinant type III collagen, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ NO.2.

[0011] The present invention also provides a recombinant expression vector comprising the nucleic acid molecule.

[0012] In the present invention, the recombinant expression vector is a pPIC9K plasmid.

[0013] The present invention also provides a recombinant yeast expressing the recombinant type III collagen.

[0014] In the present invention, the recombinant yeast uses Pichia pastoris GS115 as a host cell.

[0015] The present invention also provides a method for preparing recombinant type III collagen, comprising the following steps: inoculating a recombinant yeast expressing the above-mentioned recombinant type III collagen into a fermentation medium for fermentation culture, removing the bacteria by centrifugation to obtain a supernatant containing the recombinant type III collagen, and obtaining the recombinant type III collagen after purification.

[0016] In the present invention, the purification method is salting-out, ultrafiltration, affinity chromatography or gel filtration chromatography.

[0017] The present invention also provides the use of the recombinant type III collagen in the preparation of a product for enhancing skin regeneration ability.

[0018] In the present invention, the product is food, cosmetics or pharmaceutical product.

[0019] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0020] The reagents and raw materials used in the present invention are commercially available.

[0021] The positive effects of the present invention are: (1) the recombinant type III collagen can achieve a high protein purity (above 98%) and protein yield (above 1.2 g / L), meeting the needs of large-scale production.

[0022] (2) The yeast expression system used in the present invention has a lower risk of immune rejection and avoids the potential disease transmission risks that may be caused by traditional animal-derived collagen.

[0023] (3) Recombinant type III collagen has extremely high consistency with natural collagen and can be well integrated into skin tissue, promoting skin regeneration and repair.

[0024] (4) This technology can be applied to multiple fields such as skin repair, anti-aging, and tissue engineering, and has high economic value and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the electrophoresis diagram of the recombinant type III collagen after separation and purification.

[0026] Figure 2 This is a cross-sectional image of skin regeneration in mice in the control group.

[0027] Figure 3 This is a cross-sectional image of mouse skin regeneration in the recombinant type III collagen group. DETAILED DESCRIPTION

[0028] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0029] Example 1: Gene construction of recombinant type III collagen.

[0030] The amino acid sequence of the exon 2 to exon 4 region of the human type III collagen α1 (COL3A1) chain gene was amplified and synthesized using whole gene synthesis technology, SEQ NO.1:.

[0031]

[0032] Example 2: Gene expression of recombinant type III collagen: (1) Amplification was performed using polymerase chain reaction (PCR). Upstream primers (5'-ATG GAC GAC GAC CAA GAC G-3') and downstream primers (5'-TTA GTT GTC GACTCA GCT G-3') were added, the primer concentration was 0.5 μM, the template DNA concentration was 50 ng / μL, and the total PCR reaction system was 50 μL. The reaction conditions were set as follows: initial denaturation at 94°C for 2 minutes, followed by 30 cycles (denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute), and finally extension at 72°C for 5 minutes to ensure the integrity of the product. The PCR product was analyzed by 1% agarose gel electrophoresis, and after the expected band was observed, the product was purified. The PCR product was inserted into the expression vector pPIC9K. We first digested the pPIC9K vector with EcoRI and XhoI for double digestion at 37°C for 1 hour. Then, the digested vector was ligated with the PCR-amplified COL3A1 gene fragment using T4 DNA ligase. The reaction was carried out at 16°C overnight to ensure the efficiency and reliability of the connection. After the connection was completed, DNA sequencing was performed on the recombinant plasmid pPIC9K-COL3A1, and the success of the plasmid construction was confirmed by comparing the sequencing results with the expected sequence.

[0033] (2) Linearize the recombinant plasmid. Select Bgl II restriction enzyme as the final linearization enzyme and use it to cut the circular plasmid as follows: Add 4 μg of plasmid DNA, 4 μL of Bgl II restriction enzyme and 20 μL of enzyme digestion buffer (pH = 7) to the reaction bottle, and finally add enzyme-free water to 200 μL. The reaction was carried out at 37°C for 1 hour. Take out 5 μL of the reaction solution for agarose gel electrophoresis to detect the linearization effect. By observing the electrophoresis results, confirm whether the plasmid is successfully linearized, obtain linearized plasmid DNA, and prepare to transform it into yeast.

[0034] (3) Prepare a Yeast Peptone Dextrose Agar Plate (YPD) for streaking of Pichia pastoris GS115. Streak GS115 on a YPD plate and then place the plate in a 30°C incubator for 2 to 3 days until a single colony is clearly visible on the medium. Pick a single colony and inoculate it into a test tube containing YPD liquid culture medium. The composition of the culture medium is: 10 g / L yeast extract, 10 g / L peptone, and 20 g / L glucose. Incubate it at 30°C and 220 rpm for 24 hours to obtain a rich cell suspension. After the culture is completed, take 1 mL of the culture medium and inoculate it into two shake flasks containing fresh YPD liquid culture medium. The volume of YPD liquid culture medium in the shake flask is 50 mL. Continue to culture at 30°C and 220 rpm until the optical density (OD600) of the cells reaches 1.3 to 1.5. When OD600 reaches the predetermined value, collect the above culture solution and remove the supernatant by centrifugation at 6000 rpm for 5 minutes. Resuspend the precipitated cells in 40 mL of low-salt transformation medium and incubate in an incubator at 30°C for 30 minutes to increase the permeability of the cell membrane and prepare for electroporation transformation. After the incubation, centrifuge again at 6000 rpm for 5 minutes and remove the supernatant. Subsequently, resuspend the cells with 1 M pre-cooled sorbitol solution (Sorbitol Solution). Repeat the resuspension operation 3 times to ensure that the cells are fully washed. After the centrifugation operation is completed, add an appropriate amount of cold sorbitol solution (about 40 mL) and resuspend the cells to a final concentration of 1-2×10 8 cells / mL. At this point, the resuspended cells are competent cells. Dispense the competent cells into sterile microcentrifuge tubes, about 200 μL per tube. After dispensing, quickly store them in a -80°C refrigerator for subsequent experiments.

[0035] (4) Preparation of Pichia competent cells and electroporation. Under sterile conditions, mix 200 μL of competent cells with 10-20 μg of linear shuttle plasmid DNA and gently invert to mix, ensuring that the DNA is evenly distributed in the cell suspension. Electroporation: Transfer the mixture to a dedicated electroporation tank and set the electroporation parameters to 1.5 kV, 200 Ω, and 50 μF. Start electroporation and apply the electroporation for 5 milliseconds. During the electroporation process, holes will form in the cell membrane instantly, allowing the plasmid DNA to enter the cell. After the electroporation is completed, immediately transfer the cells in the electroporation tank to a centrifuge tube containing preheated YPD medium (volume 1 mL) and gently mix. Place the tube in an incubator at 30°C and resume the culture for 1 hour to allow the cells to have enough time to repair membrane damage and express the transferred gene. Spread the recovered cells on MD basal medium (Minimal Dextrase Medium) plates at an appropriate dilution (usually 1:10 or 1:100). The components of MD plates include: 0.67 g / L yeast extract, 0.5 g / L nitrogen source (such as amino acids or amino acid salts), 2 g / L glucose (or other carbon sources), and 15 g / L agar. Make sure the plates are evenly distributed after spreading. Place the coated MD plates in a 30°C incubator for 2-4 days until single colonies appear. By screening colonies that can grow on MD medium, the Pichia strains that have been successfully transformed are identified.

[0036] (5) Fermentation process. After picking the multi-copy transformants selected by resistance screening, they were transferred to YPD medium (yeast extract-peptone-glucose medium, composed of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 15 g / L agar) using a sterilized toothpick for activation culture. The culture conditions were 30°C, shaking speed 220 rpm, and culture time for 24 hours to promote cell growth and recovery. After the culture was completed, the activated cultured cells were taken out and inoculated into buffered glycerol-complex medium (BMGY) at a 2% inoculation rate. The ingredients included: 10 g / L yeast extract, 20 g / L peptone, 1% glycerol (v / v), and the pH was adjusted to 6.0. The culture was continued at 30°C and 220 rpm for 24 hours. Next, the cells in the BMGY medium were inoculated into the buffered methanol complex medium for induced expression of the target protein. The composition of BMMY medium is similar to that of BMGY, but methanol needs to be added as an inducer with an initial concentration of 1% (v / v) and added within the first 24 hours of culture to promote the expression of the target protein. The culture was continued for 48 hours, during which the culture conditions of 30°C and 220 rpm were maintained. After the fermentation was completed, the supernatant was collected by centrifugation at 4000 rpm for 10 minutes for subsequent protein separation and purification operations.

[0037] Example 3: Isolation and purification of recombinant type III collagen.

[0038] The fermentation broth supernatant was treated by ultrafiltration membrane, and a polysulfone membrane with a molecular weight cutoff of 10 kDa was selected to ensure that the target protein could pass through, while effectively retaining small molecules and salt ions. The operating conditions of ultrafiltration were: the operating pressure was 0.1-0.2 MPa until the liquid volume was reduced to 1 / 3 of the original volume, and the concentrated sample could be stored at 4°C for further purification. Next, the concentrated sample was subjected to cation exchange chromatography using a CM Sepharose Fast Flow column. First, the column was equilibrated with 10 volumes of elution buffer (20 mM phosphate buffer, pH 6.5). Then, the concentrate was loaded onto the column at a flow rate of 1 mL / min. The eluate was collected and the absorbance was monitored at 280 nm to identify the elution peak of the target protein. According to the intensity and characteristics of the elution peak, the corresponding eluate was pooled to ensure maximum recovery of the target protein. The collected cation exchange chromatography eluate can then be subjected to anion exchange chromatography for further purification. Select the DEAE Sepharose FastFlow column. Similarly, first equilibrate the column with elution buffer (20 mM Tris-HCl buffer, pH 8.0). Load the cation exchanged sample onto the anion exchange column, and use a gradient elution method (from 0 to 500 mM NaCl) to separate different proteins. Monitor the absorbance of the eluate at 280 nm and collect the elution peak of the target protein. The purified recombinant type III collagen was detected by 12% SDS-PAGE electrophoresis. After mixing the sample with the corresponding protein marker, heat denaturation was performed, and the electrophoresis conditions were: voltage 120 V, and electrophoresis time was 1 hour. By comparing with the standard molecular weight marker, the molecular weight and purity of the target protein were confirmed. The electrophoresis diagram of the target protein is shown below. Figure 1 As shown, the results showed that the purity of the protein reached 99.2% and the yield reached 13.8 g / L.

[0039] Example 4: Identification of recombinant type III collagen.

[0040] The identification process of recombinant type III collagen was completed by enzymatic hydrolysis and mass spectrometry analysis to confirm its amino acid sequence and purity. The specific steps are as follows: the purified recombinant type III collagen was dissolved in 50 mM amino acid buffer (pH 7.5) to ensure the smooth progress of the enzymatic hydrolysis reaction. Trypsin was added in proportion (enzyme substrate ratio of 1:100), that is, 10 μg of trypsin was added per mg of collagen, and the reaction was carried out at 37°C for 4 hours. After the first step of the reaction, trypsin was added again in the same proportion and continued to react at 37°C for 16 hours to ensure that the collagen was fully hydrolyzed. Glu-C protease was added in proportion (enzyme substrate ratio of 1:40), that is, 25 μg of Glu-C protease was added per mg of collagen, and the reaction was carried out at 37°C for 4 hours. After completing the first step, Glu-C protease was added again in the same proportion and maintained at 37°C for 16 hours to further decompose the protein. Chymotrypsin (enzyme substrate ratio of 1:40) was added, and 25 μg of chymotrypsin was added per mg of collagen, and the reaction was continued at 37°C for 4 hours. After the first step, chymotrypsin was added in the same ratio and kept at 37°C for 16 hours to ensure complete digestion of collagen. After completing the above enzymatic hydrolysis steps, the enzymatic hydrolysis products were analyzed by liquid chromatography-mass spectrometry. Chromatographic conditions: 150 μm id×150 mm capillary, filled with Acclaim PepMap RPLC C18 (particle size 3μm, pore size 100Å). Mobile phase A: 0.1% formic acid solution. Mobile phase B: 0.1% formic acid and 80% acetonitrile (ACN) mixture. The flow rate was set to 600 nL / min. Analysis time for each component: set to 66 minutes to ensure adequate separation and detection. The recombinant type III collagen was analyzed in depth by liquid chromatography-mass spectrometry, and the obtained peptides were compared with the sequence of human type III collagen α1 chain in the database using Mascot 2.0 software. The results showed that all detected peptides were completely contained in the full-length sequence of human type III collagen α1 chain, and the coverage rate was as high as 100%. This means that the recombinant protein we expressed and purified is completely consistent with the theoretical sequence, without any missing or redundant expression, which further proves the integrity and high purity of the recombinant protein.

[0041] Example 5: Control experiment of enhancing skin regeneration by recombinant type III collagen.

[0042] Animal model selection: Healthy mice (8-10 weeks old, weighing 20-25 grams) were selected as experimental subjects. Trauma model establishment: A circular skin wound with a diameter of about 4 mm was made on the back of the mouse using a sterile scalpel.

[0043] Preparation of recombinant type III collagen composition: Dissolve the purified recombinant type III collagen in physiological saline at a concentration of 8 mg / mL. Filter the solution with a 0.22 μm filter membrane to remove possible impurities. Add L-arginine (0.5 mg / mL), vitamin C (50 μg / mL), glucose (2 mg / mL) and povidone iodine (0.1 mg / mL) to the collagen solution one by one.

[0044] Prepare a control composition: Compared with the recombinant type III collagen composition, except that the recombinant type III collagen is not added, the other ingredients and concentrations are completely the same.

[0045] The experiment was carried out in two groups as follows.

[0046] (1) Control group: Use sterile gauze dipped in recombinant type III collagen composition to treat the wound surface every day. Cover the wound site with sterile dressing after each treatment to ensure that the wound surface remains clean.

[0047] (2) Experimental group: The wound surface was treated with sterile gauze dipped in the recombinant type III collagen composition every day. After one week of the experiment, the wound area of ​​each group of mice was removed and routine histological sectioning was performed. The sections were stained with HE staining to observe the cell proliferation, angiogenesis, collagen fiber deposition, etc. around the wound surface. Figure 2 and Figure 3 As shown. After using the recombinant type III collagen composition, the wound healing speed was significantly faster than that of the control group. The wound surface showed obvious healing after 2 weeks. Microscopic section observation showed that the wound surface formed tightly arranged collagen fibers, angiogenesis was relatively active, and cell proliferation was obvious. The wound surface of the control group healed slowly, and microscopic section observation showed that the collagen fibers were arranged relatively loosely and angiogenesis was not obvious. This experiment verified the significant effect of recombinant type III collagen in skin regeneration. Compared with the control group, recombinant type III collagen has significant advantages in enhancing skin regeneration ability, promoting collagen fiber deposition and wound healing. This provides an important experimental basis for the application of recombinant type III collagen prepared by the present invention as a biomedical material in skin regeneration and repair.

Claims

1. A recombinant type III collagen with enhanced skin regeneration ability, characterized in that: The amino acid sequence of the recombinant type III collagen is shown in SEQ NO.

1.

2. A nucleic acid molecule encoding the recombinant type III collagen for enhancing skin regeneration ability as claimed in claim 1.

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

2.

4. A recombinant expression vector containing the nucleic acid molecule according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector is a pPIC9K plasmid.

6. A recombinant yeast expressing the recombinant type III collagen having the ability to enhance skin regeneration as claimed in claim 1.

7. The recombinant yeast according to claim 6, characterized in that The recombinant yeast uses Pichia pastoris GS115 as a host cell.

8. A method for preparing recombinant type III collagen with enhanced skin regeneration ability, characterized in that: The recombinant yeast expressing the recombinant type III collagen with enhanced skin regeneration ability as claimed in claim 1 is inoculated into a fermentation medium for fermentation culture, the bacteria are removed by centrifugation to obtain a supernatant containing the recombinant type III collagen, and the recombinant type III collagen is obtained after purification.

9. The preparation method according to claim 8, characterized in that: The purification method is salting out, ultrafiltration, affinity chromatography or gel filtration chromatography.

10. Use of the recombinant type III collagen with skin regeneration enhancement ability as claimed in claim 1 in the preparation of cosmetics.

Images