Recombinant collagen and use thereof for preparing gels

Abstract

This invention discloses a recombinant collagen protein and its application in preparing gels, belonging to the field of bioengineering technology. The protein sequence is SEQ ID NO:3, fused with a human type III collagen α1 chain Gly-X-Y repeat region and a type II collagen α1 chain core functional region, and its natural triple helix structure was obtained through expression and purification in Pichia pastoris. In vitro experiments showed that CoF significantly promoted the proliferation of human dermal fibroblasts without cytotoxicity; in a rat full-thickness skin defect model, the CoF treatment group showed faster healing than the natural type I collagen control group. This invention also provides the application of CoF in preparing medical gels, the resulting gels exhibiting excellent biocompatibility and pro-repair activity, suitable for skin dressings, tissue engineering scaffolds, etc. This invention solves the problem of limited single collagen function, providing high-performance recombinant collagen raw materials for wound repair.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a recombinant collagen and its application in preparing a gel. Background Technology

[0002] Collagen is the most abundant structural protein in mammals, widely distributed in connective tissues such as skin, tendons, and bones. Its main functions include maintaining tissue mechanical strength, supporting cell adhesion and migration, and participating in signal transduction. Traditionally, collagen used in medicine is mostly extracted from animal tissues (such as bovine Achilles tendons and pig skin). While the sources are wide-ranging, these sources present risks such as immunogenicity, pathogen residues, and batch-to-batch quality variations, limiting its application in high-end medical devices and regenerative medicine.

[0003] In recent years, recombinant collagen technology has developed rapidly. By expressing human collagen sequences in microbial or mammalian cells through genetic engineering, immunogenicity can be significantly reduced and purity improved. Current research largely focuses on the recombinant expression of single collagen types (such as type I or type III only), but it still falls short in mimicking the multifunctional synergistic effects of the natural extracellular matrix (ECM). For example, type III collagen is involved in early wound healing and flexibility regulation, type II collagen plays a prominent role in cartilage repair, and type I collagen provides mechanical support; using only one type is insufficient to comprehensively cover complex repair needs. Furthermore, the thermal stability and solubility of recombinant collagen still differ from natural collagen, affecting its processing adaptability in gels, dressings, and other dosage forms. Therefore, developing a recombinant collagen protein that integrates the advantages of multiple collagen types, possesses both high bioactivity and processing adaptability, and expanding its application in medical gel formulations is of great significance for improving the overall performance of wound repair materials. Summary of the Invention

[0004] The present invention aims to solve the problems of limited bioactivity, single function and insufficient dosage form adaptability of existing recombinant collagen, and provides a recombinant collagen and its application in preparing gels.

[0005] To achieve the above objectives, the present invention first provides a recombinant collagen protein, the amino acid sequence of which is shown in SEQ ID NO:3.

[0006] The present invention also provides a nucleotide molecule encoding the above-mentioned recombinant collagen, said nucleotide molecule being shown in SEQ ID NO:4.

[0007] The present invention also provides a recombinant expression vector comprising the above-described nucleotide molecules.

[0008] In some embodiments, the recombinant expression vector is a pPICZαA-CoF vector capable of expression in yeast.

[0009] The present invention also provides a genetically engineered bacterium containing the above-mentioned recombinant expression vector, wherein the genetically engineered bacterium is Pichia pastoris strain X33.

[0010] The present invention also provides a method for preparing the above-mentioned recombinant collagen, comprising the following steps: (1) Cultivate the above-mentioned genetically engineered bacteria, (2) Expression of recombinant collagen under methanol-induced conditions. (3) The recombinant collagen is obtained by separating and purifying it from the fermentation supernatant or lysate.

[0011] The present invention also provides the use of the above-described recombinant collagen in the preparation of compositions for promoting cell proliferation.

[0012] In some embodiments, the cells are human dermal fibroblasts.

[0013] The present invention also provides the use of the above-mentioned recombinant collagen in the preparation of medical preparations for skin wound repair.

[0014] Finally, the present invention provides a medical gel comprising an effective amount of the above-mentioned recombinant collagen and a pharmaceutically acceptable gel matrix.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) By integrating the functional areas of type III and type II collagen, the early healing concept of type III and the tissue remodeling function of type II are integrated, overcoming the limitations of single collagen bioactivity; (2) The Pichia pastoris expression system has high yield and low cost. P4H co-expression ensures hydroxylation modification and maintains triple helix stability; (3) It contains no animal-derived components, has low immunogenicity, and is safer than traditionally extracted collagen; (4) Its physicochemical properties are suitable for processing into gel and other dosage forms, and it has clear application value in wound repair and tissue engineering. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the SDS-PAGE electrophoresis results of recombinant collagen CoF in Example 2 of this invention. In the figure: M is the protein molecular weight standard; lane 1 is CoF protein under non-reducing conditions; lane 2 is CoF monomer under reducing conditions.

[0017] Figure 2 This is a bar chart showing the effect of different concentrations of CoF on the proliferation of human dermal fibroblasts (HDF) in Example 3 of this invention. The horizontal axis represents the culture time (day 1, day 3, day 5, day 7), and the vertical axis represents the relative proliferation rate (RGR) (%).

[0018] Figure 3This is a schematic diagram of the wound healing process of CoF in a rat skin full-thickness defect model in Example 4 of the present invention. Detailed Implementation

[0019] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0020] Example 1: Sequence design of recombinant collagen The amino acid sequences of human type II collagen α1 chain (COL2A1) and human type III collagen α1 chain (COL3A1) were obtained from UniProt (https: / / www.uniprot.org / uniprotkb?query=collagen). Based on the triple helix structure of human type III collagen α1 chain, a design was developed to maximize the preservation of the function of natural human collagen. The amino acid sequence of type III collagen (SEQ ID No. 1: AGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPK) and the amino acid sequence of type II collagen (SEQ ID No. 1: 1253-1487) were selected. No. 2: AEVDATLKSLNNQIESIRSPEGSRKNPARTCRDLKLCHPEWKSGDYWIDPNQGCTLDAMKVFCNMETGETCVYPNPANVPKKNWWSSKSKEKKHIWFGETINGGFHFSYGDDNLAPNTANVQMTFLRLLSTEGSQNITYHCKNSIAYLDEAAGNLKKALLIQGSNDVEIRAEGNSRFTYTALKDGCTKHTGKWGKTVIEYRSQKTSRLPIIDIAPMDIGGPEQEFGVDIGPVCFL) was designed as the core functional region element, and the recombinant protein was designed with 6 Starting with the His tag, it is followed by a TEV restriction site to facilitate subsequent purification and digestion. Then, type I collagen and type II collagen are sequentially linked to obtain a new recombinant collagen, which is named CoF. The complete amino acid sequence of CoF is SEQ ID No.3: MGSSHHHHHHSSENLYFQGAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKAEVDATLKSLNNQIESIRSPEGSRKNPARTCRDLKLCHP EWKSGDYWIDPNQGCTLDAMKVFCNMETGETCVYPNPANVPKKNWWSSKSKEKKHIWFGETINGGFHFSYGDDNLAPNTANVQMTFLRLSTEGSQNITYHCKNSIAYLDEAAGNLKKALLIQGSNDVEIRAEGNSRFTYTALKDGCTKHTGKWGKTVIEYRSQKTSRLPIIDIAPMDIGGPEQEFGVDIGPVCFL.

[0021]

[0022] Example 2: Expression and purification of CoF in Pichia pastoris Gene synthesis was commissioned to Suzhou Genewiz Technology Co., Ltd., resulting in the recombinant expression vector pPICZαA-CoF. This vector was then transformed into DH5α to extract plasmids. The plasmids were linearized using SacI digestion for subsequent electroporation. Similarly, the pPIC9K-P4H plasmid was linearized using SacI digestion. Pichia pastoris X33 was activated using YPD plates and cultured at 30°C for 2–3 days. Single colonies were picked and transferred to 5 mL of YPD liquid medium and cultured overnight at 30°C and 220 rpm. The following day, 1 mL of the culture was transferred to 100 mL of YPD liquid medium and cultured until OD500. 595 The culture flask was placed on ice for 15 min, centrifuged at 1500g for 5 min at 4℃ to collect the bacterial cells, resuspended in 10 mL of pre-chilled sterile water at 4℃, and centrifuged again. The bacterial cells were washed twice with sterile water, then twice with pre-chilled 1M sorbitol, centrifuged at 1500g for 5 min at 4℃ to collect the cells, resuspended in 200 μL of 1M sorbitol, and aliquoted into 1.5 mL centrifuge tubes (100 μL / tube) and placed on ice. Electroporation was then immediately performed. 10 μg of linearized pPICZαA-CoF plasmid was added to X33 competent cells, gently mixed, and placed on ice for 5 min. Electroporation was performed using a 2 mm electroporator cuvette with parameters set to 1.5 kV, 200 Ω, 25 μF, and a pulse time of approximately 5 ms. Immediately after electroporation, add 1 mL of frozen 1M sorbitol, incubate at 30°C for 1 hour, and plate onto YPD plates containing 100 μg / mL Zeocin. Incubate at 30°C for 3–4 days until clear single colonies are visible. Pick single colonies for PCR using primers GACTGGTTCCAATTGACAAGC (SEQ ID No. 5) and GCAAATGGCATTCTGACATCC (SEQ ID No. 6). Select positive clones for expansion culture and prepare competent cells according to the aforementioned steps. Electroporate linearized pPIC9K-P4H, plate onto YPD plates containing 0.5 mg / mL G418, and scrape off colonies after growth. Plate onto YPD plates containing 1 mg / mL G418 to screen for high-expression strains. Pick single colonies and transfer to 5 mL of BMGY, incubate overnight at 30°C and 220 rpm. The next day, transfer to 50 mL of BMGY and incubate until OD. 595 Collect bacterial cells by centrifugation at approximately 4.4°C and 1500g for 10 minutes, then resuspend in BMMY to OD. 595The protein was induced at 20°C and 200 rpm for 96 h using α-ketoglutarate (1 mM), FeSO4 (0.1 mM), and ascorbic acid (5 mM) as inducers, with methanol added every 24 h. The protein was centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was filtered through a 0.22 μm filter. The protease inhibitors PMSF (1 mM) and EDTA (5 mM) were added. Affinity chromatography was performed using a nickel column, eluting impurities with 30 mM imidazole and proteins with 250 mM imidazole. The eluted protein was concentrated using Amicon ultrafiltration tubes, and the buffer was replaced with DPBS. 3 μg of CoF protein was added to a loading buffer containing DTT and boiled for denaturation. Another 3 μg of CoF protein was added to a loading buffer without DTT and analyzed by SDS-PAGE. The results are shown below. Figure 1 As shown. M is the molecular weight marker for the protein. Lane 1 is the non-reduced triple helix structure with a molecular weight of about 120 kD. Lane 2 is the monomer depolymerized in the reduced state with a molecular weight of about 41 kD, which is close to the theoretical value. The band is single and clear, without any extraneous bands.

[0023] Example 3: Evaluation of the promoting effect of CoF on cell proliferation Human dermal fibroblasts (HDF) were resuscitated and passaged using DMEM + 10% FBS + 1% PS. Cells in the logarithmic growth phase were harvested, the culture medium was discarded, and the cells were washed once with DPBS. Trypsin was added for 1-2 minutes to digest the cells, and complete culture medium was added to terminate the digestion. The cell suspension density was then adjusted to 5. 10 4 Cells / mL. A total of 4 96-well plates were prepared and labeled day1 / 3 / 5 / 7. 100 μL of cell suspension was added to each well of each 96-well cell culture plate, and the mixture was thoroughly mixed before incubation at 37°C with 5% CO2.

[0024] After 24 hours of culture, cells were observed under a microscope to confirm good adherence. The cell culture medium was discarded, and 100 μL / well of CoF protein diluent was added. The concentrations of the CoF protein diluent were 10 μg / mL, 50 μg / mL, and 100 μg / mL. The blank control group was not seeded with cells, containing only 100 μL of complete culture medium; the negative control group was seeded normally, but with 100 μL of complete culture medium added. The positive control group contained 100 μL of complete culture medium containing 1 mg / mL of natural human type I collagen (Abcam, catalog number ab7533). Each group had 5 replicates. The drug addition diary was recorded as day 0, and 24 hours later was day 1.

[0025] On days 1, 3, 5, and 7, the correspondingly labeled cell culture plates were removed, and 10 μL of CCK-8 was added to each well. After thorough mixing, the plates were incubated at 37°C for 1.5 h. The absorbance (A) at 450 nm was measured using a microplate reader, and the relative cell proliferation rate (RGR) was calculated as follows: RGR(%) = (Absorbance of experimental group - Absorbance of blank group) / (Absorbance of negative control group - Absorbance of blank group) × 100%. CoF toxicity was evaluated based on the relative cell proliferation rate. RGR(%) ≥ 100 indicated no cytotoxicity; RGR(%) 75-99 represented very low cytotoxicity; RGR(%) 50-74 represented low cytotoxicity; RGR(%) 25-49 represented moderate cytotoxicity; RGR(%) 1-24 represented high cytotoxicity; and RGR(%) = 0 represented very high cytotoxicity or lethality. The results of the CoF effect on HDF proliferation rate determination are as follows: Figure 2 As shown, the RGR (%) at each concentration was greater than or equal to 100, and the increase in RGR (%) at the same time was dose-dependent. At the same concentration, the proliferation promotion of HDF by CoF increased with time, while no cytotoxicity was observed at high concentrations of CoF, indicating good safety.

[0026] Example 4: Evaluation of the therapeutic effect of CoF in a rat model of full-thickness skin defects Eighteen healthy and active SD rats were randomly divided into three groups of six each. After anesthesia, the rats were disinfected with povidone-iodine, and the skin was prepared. A disposable sterile skin harvester (8 mm in diameter) was used to harvest full-thickness skin from the central back, down to the fascia layer. After wound cleaning, treatment was administered according to Table 1, once daily. The wound was secured with sterile gauze after each administration. A surgical diary was kept as day 0. Postoperatively, the gauze was changed daily, and the wound diameter was measured and recorded. Wound healing and scar formation were observed and recorded. Figure 3 Regarding wound healing, compared with the same dose of natural human type I collagen, the CoF treatment group showed a comparable wound healing speed in the early stages of treatment. Starting from day 5 of administration, the wounds in the CoF treatment group healed faster than those in the positive control group. CoF has good application value and potential in wound repair.

[0027] Table 1. Dosing Grouping

[0028] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

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

3.

2. A nucleotide molecule encoding the recombinant collagen of claim 1, characterized in that, The nucleotide molecule is shown in SEQ ID NO:

4.

3. A recombinant expression vector comprising the nucleotide molecule of claim 2.

4. The recombinant expression vector according to claim 3, characterized in that, The recombinant expression vector is the pPICZαA-CoF vector, which can be expressed in yeast.

5. A genetically engineered bacterium, characterized in that, The compound contains the recombinant expression vector as described in claim 3 or 4, wherein the genetically engineered bacterium is Pichia pastoris strain X33.

6. A method for preparing the recombinant collagen according to claim 1, characterized in that, Includes the following steps: (1) Cultivating the genetically engineered bacteria as described in claim 5, (2) Expression of recombinant collagen under methanol-induced conditions. (3) The recombinant collagen is obtained by separating and purifying it from the fermentation supernatant or lysate.

7. Use of the recombinant collagen of claim 1 in the preparation of a composition for promoting cell proliferation.

8. The application according to claim 7, characterized in that, The cells are human dermal fibroblasts.

9. The use of the recombinant collagen of claim 1 in the preparation of a medical preparation for skin wound repair.

10. A medical gel, characterized in that, It comprises an effective amount of the recombinant collagen of claim 1, and a pharmaceutically acceptable gel matrix.

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