Recombinant collagen III and application thereof in preparation of gel

Abstract

The invention relates to the technical field of biology, and particularly discloses a recombinant collagen III and application thereof in preparation of gel. The recombinant collagen III is designed by optimizing functional area sequences of human I-type and III-type collagen, the amino acid sequence is shown as SEQ ID No.2, and the recombinant collagen III has the characteristics of high stability, good hydrophilicity and low immunogenicity. The preparation method comprises the steps of expression vector construction, escherichia coli induced expression, affinity chromatography purification and renaturation. The recombinant collagen III can be prepared into a gel dressing and comprises sodium alginate, methylparaben and other components. Experiments show that the gel can effectively promote cell proliferation and has no cytotoxicity; in a mouse skin injury model, the collagen can relieve inflammation, accelerate wound healing and inhibit scar formation, and the effect of the collagen is superior to that of natural human III-type collagen. The invention provides a safe and efficient novel material for wound repair, and is suitable for medical dressings and tissue engineering.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering and medical biomaterials, specifically relating to a recombinant collagen III and its application in preparing a gel. Background Technology

[0002] Collagen is the main structural protein of animal connective tissue, accounting for 25%-30% of the total protein in the human body. It is widely distributed in the skin, bones, and blood vessels, and has functions of support, repair, and biosignal transduction. Type I and Type III collagen are the two most abundant types in the skin. Type III collagen plays a key role in early embryonic development and wound healing, promoting cell migration and proliferation and reducing scar formation. However, natural collagen has many limitations: First, natural collagen extracted from animals or humans may carry pathogens (such as viruses or prions), triggering immune responses and increasing the risk of disease transmission; second, natural collagen has poor solubility and low stability, is easily denatured and inactivated during processing, and its isoelectric point often deviates from the physiological range (e.g., human type III collagen has a slightly acidic pI), leading to easy aggregation or degradation during in vivo application; in addition, the production of natural collagen relies on tissue extraction, which is costly, time-consuming, and has large batch-to-batch variations, making it difficult to meet the needs of large-scale clinical applications.

[0003] In existing technologies, recombinant collagen is prepared through genetic engineering, which can reduce immunogenicity and improve purity. For example, some studies have developed recombinant sequences based on type I collagen, but these mainly focus on bone repair applications and have poor effects on skin wound healing. Furthermore, the sequence design is not optimized for isoelectric point, resulting in insufficient protein stability under physiological conditions. Other studies use short peptide fragments of type III collagen, which improves solubility but has weak bioactivity and cannot mimic the functional region interactions of the full-length protein, limiting its ability to promote cell proliferation and tissue regeneration. In recent years, researchers have attempted to fuse the functional regions of type I and type III collagen to balance mechanical strength and elasticity, but sequence fusion often neglects the balance between hydrophilicity and immunogenicity. For example, some recombinant proteins have a predominantly alkaline pI (e.g., pI>9), which may interfere with the cellular microenvironment pH and trigger inflammatory responses. In the field of gel dressings, natural polysaccharides such as sodium alginate are often used as carriers, but simple polysaccharide dressings lack bioactivity and have slow healing speeds. Adding collagen can enhance the repair effect, but existing collagen gels are mostly based on natural proteins, which can easily lead to increased exudate and scar formation. While existing market products such as natural human type III collagen gel have shown some effectiveness, animal studies have revealed healing cycles exceeding 20 days, and they are also costly. Therefore, there is an urgent need to develop a recombinant collagen type III gel with a near-neutral pI, high stability, and the ability to be mass-produced.

[0004] This invention addresses the aforementioned problems by fusing the functional region sequences of type I and type III collagen through bioinformatics optimization to obtain a novel recombinant collagen III (Fus2) with a pI of 7.26, close to the physiological environment, and increased hydrophilicity. Experiments have demonstrated that this protein is non-cytotoxic and can accelerate wound healing and inhibit scarring, filling a gap in existing technologies. Summary of the Invention

[0005] The present invention first provides a recombinant collagen III, comprising an optimized combination of functional region sequences of human type I and type III collagen, the amino acid sequence of which is shown in SEQ ID No. 2.

[0006] The present invention also provides a nucleotide sequence encoding the recombinant collagen III of claim 1.

[0007] In some embodiments, the nucleotide sequence shown is as shown in SEQ ID No. 3.

[0008] The present invention also provides an expression vector comprising the above-described nucleotide sequence and having a His tag, for expressing recombinant collagen III in a prokaryotic host.

[0009] The present invention also provides a host cell, namely Escherichia coli BL21, containing the above-mentioned expression vector, for inducing expression of recombinant collagen III.

[0010] The present invention also provides a method for preparing the above-mentioned recombinant collagen III, comprising the following steps: transforming the expression vector into host cells, inducing expression with IPTG, collecting bacterial cells by centrifugation, sonicating, purifying by Ni2+-NTA affinity chromatography, and refolding and dialysis of the protein.

[0011] The present invention also provides a gel comprising the above-mentioned recombinant collagen III, sodium alginate, methylparaben, and buffer solution.

[0012] In some embodiments, the concentration of recombinant collagen III is 1.5 g / L, sodium alginate is 18.2 g / L, methylparaben is 0.25 g / L, and the buffer solution is DPBS at pH 7.4.

[0013] The present invention also provides the use of the above-mentioned gel in the preparation of drugs or medical dressings that promote wound healing.

[0014] The present invention also provides the application of the above-mentioned gel in inhibiting scar formation.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Low immunogenicity and high safety: After sequence optimization, the pI is 7.26, which is close to neutral and reduces the rejection reaction in vivo; MTT assay showed that the relative cell proliferation rate (RGR) at each concentration was ≥100%, and there was no cytotoxicity.

[0016] (2) Promotes cell proliferation: The concentration-dependent effect is significant, with the highest RGR at 3 mg / mL, which is superior to the natural type III collagen control.

[0017] (3) Accelerate wound healing: In animal experiments, the experimental group (Fus2 gel) wounds healed in 18 days, the control group (natural collagen) wounds healed in 20 days, and the model group healed in 24 days with scarring, indicating that Fus2 gel can reduce inflammation and promote repair.

[0018] (4) Inhibition of scar formation: The experimental group had no scars after healing, while the model group had obvious scars, highlighting its cosmetic and clinical value.

[0019] (5) Simple process and low cost: The E. coli expression system is easy to scale up and produce. The gel preparation only requires stirring and mixing, which is suitable for industrialization. Attached Figure Description

[0020] Figure 1 This is an SDS-PAGE analysis of the recombinant Fus2 protein.

[0021] Figure 2 This is a diagram showing the results of an experiment on how Fus2 promotes the proliferation of L929 cells.

[0022] Figure 3 This is a diagram showing the therapeutic effect of a mouse skin lesion model. Detailed Implementation

[0023] 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.

[0024] Example 1: Sequence design of humanized recombinant collagen III Human type I collagen α1 chain (GenBank: KAI2583922.1; collagen type I alpha 1 chain [Homo sapiens]) and human type III collagen α1 chain (GenBank: CSS32583.1; prepro-alpha-1 type 3 collagen [Homo sapiens]) were obtained from NCBI (https: / / www.ncbi.nlm.nih.gov / ). Based on the collagen sequences, optimization was performed while preserving functional regions to reduce immunogenicity and increase solubility. Amino acids 230-418 of the functional region sequence of type I collagen and amino acids 404-651 and 836-917 of the functional region sequence of type III collagen were selected and joined end-to-end in a III+I+III sequence. The ends were modified with amino acids 1078-1107 of type I collagen to obtain a new recombinant type III collagen amino acid sequence, SEQ ID No. 1, named Fus1. No.1:PGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMR GMPGSPGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGGDDGEAGKPGRPGERGPPGPQGAR GLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGAD GQPGAKGANGAPGIAGAPGFPGARGPSGPPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPVGPVGARGPAGPQGPRGDKGETGEQGDRGI Analysis of the Fus1 sequence using the proteomics database (https: / / web.expasy.org / protparam / ) revealed a theoretical pI of 9.75, indicating a slightly basic nature. It consists of 549 amino acids with a relative molecular weight of 48811.96, an instability coefficient of 23.61 (relatively stable), and a total hydrophilicity coefficient of -0.934. Based on these indicators, the Fus1 sequence was optimized to obtain the amino acid sequence Fus2 (SEQ ID No. 2: Analysis of Fus2 revealed a theoretical pI of 7.26, indicating a slightly neutral protein composition. It consists of 549 amino acids with a relative molecular weight of 48582.07, an instability coefficient of 22.73, and a total hydrophilicity coefficient of -1.010. Compared to Fus1, Fus2 has a more neutral pI, almost no change in protein stability, and increased hydrophilicity. Codon optimization of the Fus2 sequence without altering the amino acid sequence yielded the nucleotide sequence SEQ ID No. 3 (SEQ ID No. 3:

[0025] Example 2: Construction of Recombinant Expression Vector Gene synthesis was commissioned to Suzhou Genewiz Technology Co., Ltd., resulting in a recombinant expression vector with a His tag, named pET-28a-Fus2, which was then transformed into BL21 *E. coli*. The specific steps were as follows: 2 μL of plasmid was added to 50 μL of competent BL21 *E. coli* cells and incubated on ice for 30 min; heat-shocked at 42℃ for 90 s, followed by immediate ice incubation for 2 min; 500 μL of antibiotic-free LB medium was added, and the cells were incubated at 37℃ and 220 rpm for 1 h; 100 μL of the bacterial culture was spread onto LB agar plates containing Kan (final concentration 50 μg / mL) and incubated upside down at 37℃ for 12–16 h. Single clones were picked for sequencing, and after confirming accurate sequencing, Fus2 protein expression was induced. Recombinant pET-28a-Fus2 bacteria were grown to the logarithmic growth phase in a shaker, then IPTG was added to a final concentration of 0.5 mmol / L, and the bacteria were induced and cultured at 37°C for 12 h. 200 mL of the induced recombinant bacteria were collected by centrifugation, resuspended in PBS, and incubated on ice for 30 min. The bacteria were then sonicated, and the supernatant was collected by centrifugation. The precipitate was dissolved in 10 mL of inclusion body denaturation solution containing 20 mmol / L disodium hydrogen phosphate, 20 mmol / L sodium dihydrogen phosphate, 0.5 mmol / L sodium chloride, and 8 mmol / L urea. The supernatant was then collected by centrifugation and chelated with a Ni2+-NTA affinity chromatography column overnight at 4°C. The following day, gradient elution was performed using elution buffers containing 20, 50, 100, 300, and 500 mmol / L imidazole. The eluent was collected stepwise, and the purified protein was refolded at 4°C for 24 hours using protein refolding working solution. The dialysis bag was rinsed, soaked, and leak-tested to ensure no leakage. The protein sample to be dialyzed was slowly injected into the dialysis bag, and the bag opening was clamped tightly with dialysis clamps to ensure a tight seal. 50 times the volume of PBS buffer was added to a beaker, and the sealed dialysis bag was completely immersed in the PBS buffer, ensuring the sample was completely covered. Dialysis was performed with magnetic stirring at 4°C. After dialysis for 2–4 hours, fresh PBS was replaced, and dialysis continued overnight. The dialysis product was further purified using molecular sieves (Superdex 75 prep grade packing material). 3 μg of the purified recombinant Fus2 protein was analyzed by SDS-PAGE. See [link to SDS-PAGE analysis]. Figure 1 Lane 1 contains recombinant Fus2, with a single band and no other molecular weight impurities. The molecular weight is around 50 kDa, which is close to the molecular weight theoretically calculated in Example 1.

[0026] Example 3: Evaluation of Fus2's role in promoting cell proliferation Mouse fibroblast L929 cells were cultured in DMEM + 10% FBS medium at 37°C and 5% CO2 until the logarithmic growth phase, reaching a cell density of approximately 80% and a cell viability of over 95%. The digested cells were resuspended in medium to a density of 5000 cells / mL, and 100 μL / well was added to each well of a 96-well plate. After 24 hours of culture, the cells were observed under a microscope to confirm good adherence. The cell culture medium was discarded, and 100 μL / well of Fus2 protein dilution buffer was added. The Fus2 protein dilution buffer was prepared using DMEM medium, with concentrations of 1 mg / mL, 2 mg / mL, and 3 mg / mL. The negative control group received an equal volume of DMEM medium without protein, while the positive control group (denoted as positive) received an equal volume of natural human type III collagen (Merck, catalog number cc054) containing 1 mg / mL. Each group had three replicates. After 48 hours of protein treatment, 50 μL of MTT was added to each well, and the cells were cultured at 37°C for another 2 hours. The original culture was then discarded, and 150 μL of DMSO was immediately added to each well. The cells were incubated at room temperature with gentle shaking for 20 minutes. The absorbance (A) at 490 nm was measured using a microplate reader, and the relative cell proliferation rate (RGR) was calculated as (absorbance of experimental group / absorbance of negative control group) × 100%. The cytotoxicity of recombinant collagen Fus2 was evaluated based on the relative cell proliferation rate. An RGR ≥ 100 indicated no cytotoxicity; an RGR of 75-99 represented very low cytotoxicity; an RGR of 50-74 represented low cytotoxicity; an RGR of 25-49 represented moderate cytotoxicity; an RGR of 1-24 represented high cytotoxicity; and an RGR of 0 represented very high cytotoxicity or lethality. The results of the relative cell proliferation rate measurement of Fus2 after 48 hours are shown below. Figure 2 As shown, the RGR (%) at each concentration was greater than or equal to 100, and the RGR (%) increased slowly with increasing concentration, indicating that the cell proliferation-promoting effect of Fus2 is concentration-dependent. At the same time, Fus2 has no cytotoxicity and good safety.

[0027] Example 4: Preparation of Fus2 Gel Weigh 18.2g of sodium alginate, 1.5g of Fus2 protein, and 0.25g of methylparaben, place them in a mixing container, add DPBS buffer (pH 7.4) to 1000ml, and stir to dissolve at 1500 rpm for 2 hours. Centrifuge at 2000 rpm at room temperature for 1.5 hours to remove foam. After sterilization, aliquot and store. Prepare natural human type III collagen gel using the same method as a control for subsequent animal experiments.

[0028] Example 5: Evaluation of the therapeutic effect of Fus2 in a mouse skin lesion model Healthy and active Balb / c-Nude mice were selected, anesthetized, and disinfected with povidone-iodine before skin preparation. Two full-thickness skin grafts were harvested from the central part of the mouse's back using a disposable sterile dermatome (8mm diameter) to create a full-thickness skin defect model. The wounds were cleaned. For the experimental group, Fus2 gel dressing was applied to the left lesion, and medical gauze cut to the appropriate size and shape was applied to the wound. A suitable length of medical bandage was then carefully wrapped around the wound, avoiding excessive tightness that could impede blood flow. The right lesion served as the control group, receiving natural human type III collagen gel dressing, with other treatments the same as the experimental group. A model group was also established; neither wound in the model group received gel, and other treatments were the same as the experimental group. The surgical diary was recorded as day 0, with gauze changed daily post-surgery. Gel was reapplied, wound diameter was measured and recorded, and wound healing and scar formation were observed and recorded. Changes in wound area were recorded as follows. Figure 3 As shown, on day 1, there was no significant difference in wound area between the experimental group and the control group. Both groups had a small amount of exudate and redness and swelling at the wound edges. The model group had more exudate and significant redness and swelling at the wound edges and base. In the experimental group, the wound began to shrink on day 4, with no exudate, and healed by day 18 without scarring. In the control group, the wound exudate decreased on day 4, and the edges remained red and swollen. The wound area began to shrink on day 6, and healed by day 20 without scarring. In the model group, the wound began to shrink on day 8, and significant scarring was visible on day 24. Therefore, Fus2 gel can reduce inflammation, promote wound repair and healing, and inhibit scar formation. Compared with existing natural human type III collagen, Fus2 has a better effect on promoting wound healing.

[0029] 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 III, characterized in that, An optimized combination of functional region sequences containing human type I and type III collagen, the amino acid sequence of which is shown in SEQ ID No.

2.

2. A nucleotide sequence encoding the recombinant collagen III of claim 1.

3. The nucleotide sequence according to claim 2, characterized in that, The nucleotide sequence shown is as shown in SEQ ID No.

3.

4. An expression carrier, characterized in that, It comprises the nucleotide sequence of any one of claims 2-3 and is tagged with His, for expressing recombinant collagen III in a prokaryotic host.

5. A host cell, characterized in that, It is Escherichia coli BL21, containing the expression vector of claim 4, for inducing expression of recombinant collagen III.

6. A method for preparing the recombinant collagen III of claim 1, characterized in that, The process includes the following steps: transforming the expression vector into host cells, inducing expression with IPTG, collecting bacterial cells by centrifugation, sonicating, purifying by Ni2+-NTA affinity chromatography, and refolding and dialysis of the protein.

7. A gel, characterized in that, It comprises the recombinant collagen III as described in claim 1, as well as sodium alginate, methylparaben, and buffer solution.

8. The gel according to claim 7, characterized in that, The concentration of the recombinant collagen III was 1.5 g / L, sodium alginate was 18.2 g / L, methylparaben was 0.25 g / L, and the buffer solution was DPBS at pH 7.

4.

9. The use of the gel according to claim 7 or 8 in the preparation of a medicament or medical dressing that promotes wound healing.

10. The use of the gel according to claim 7 or 8 in inhibiting scar formation.

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