Recombinant protein for promoting burn repair and application thereof

Abstract

The application relates to the fields of genetic engineering and biological medicine, and particularly discloses a recombinant protein for promoting burn repair and application thereof. The recombinant protein comprises an improved human antibacterial peptide LL-37 and an improved human epidermal growth factor hEGF, and the two are connected through a flexible linker. In the amino acid sequence of the LL-37, the 26th Asp is replaced by Asn; in the amino acid sequence of the hEGF, the 41st Arg is replaced by Lys, and the 47th Leu is replaced by Val. Experiments show that the recombinant protein retains excellent antibacterial activity and efficient cell proliferation function, and shows more significant effect of promoting burn wound healing than single-component proteins in an in-vivo animal model. The application also relates to a pharmaceutical composition containing the recombinant protein and application of the pharmaceutical composition in preparation of a medicine for treating burns and promoting wound repair.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biomedicine, and more specifically, to a recombinant protein that promotes burn repair and its applications. Background Technology

[0002] Burns are a common injury to the skin and subcutaneous tissue, usually caused by factors such as heat, chemicals, electric current, or radiation. Severe burns, especially full-thickness burns, can lead to complete destruction of the epidermis and dermis, creating open wounds that are prone to secondary bacterial infections. They also severely impair the skin's barrier, regeneration, and immune functions, causing immense suffering and potentially leading to systemic inflammatory response syndrome, organ failure, and even death. Therefore, promoting rapid wound healing and restoring skin integrity are core goals of burn treatment.

[0003] An ideal burn wound treatment strategy needs to address several key issues simultaneously. First, controlling infection is crucial. Open wounds are breeding grounds for bacteria; infection exacerbates local inflammation, leading to further tissue necrosis and severely hindering the healing process. Second, it is essential to effectively promote the proliferation, migration, and differentiation of skin cells to achieve re-epithelialization and tissue regeneration. Among numerous bioactive factors, human epidermal growth factor (HGF) has been widely used in the repair and treatment of burns, chronic ulcers, and other wounds due to its powerful ability to promote the proliferation of epidermal cells and fibroblasts. However, single-component HGF preparations lack antibacterial activity; their healing-promoting effect is significantly reduced, or even rendered ineffective, on wounds at risk of infection or already infected.

[0004] Antimicrobial peptides are an important component of the body's innate immune defense system. Among them, the human antimicrobial peptide LL-37 not only exhibits broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria, but also possesses immunomodulatory, angiogenesis-promoting, and wound-healing effects. However, LL-37 alone is easily degraded by proteases under physiological conditions, exhibiting limited stability, and its direct stimulatory effect on cell proliferation is relatively weak, unable to completely replace the core functions of growth factors. Therefore, in clinical practice, it is often necessary to use antimicrobial drugs in combination or sequentially with growth factor drugs, which increases the complexity of treatment regimens, the potential risk of drug interactions, and the economic burden on patients.

[0005] In existing technologies, studies have attempted to fuse antimicrobial peptides with growth factors to obtain single molecular entities with both antibacterial and healing-promoting functions. For example, LL-37 has been linked to a vascular endothelial growth factor fragment. However, the design of such fusion proteins faces numerous challenges. Simple physical mixing or random linking may disrupt the original active conformations of each functional domain, leading to loss or weakening of function. Furthermore, the increased molecular weight and structural changes of the fused protein may affect its tissue permeability, in vivo stability, and safety. More importantly, the key to the successful development of such multifunctional fusion proteins lies in achieving efficient fusion of functional domains through precise molecular design while ensuring that each domain independently and fully exerts its biological effect.

[0006] Currently, there are no mature products on the market that simultaneously integrate highly effective antibacterial and potent epidermal cell proliferation-promoting functions, and have undergone rigorous molecular design and validation. Therefore, developing a novel recombinant fusion protein that can synergistically promote burn wound healing is of great significance for simplifying treatment plans, improving efficacy, and reducing infection risks, and has broad clinical application prospects and market value. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a single recombinant protein molecule that can simultaneously and effectively inhibit bacterial infection of wounds and strongly promote cell proliferation and tissue repair, so as to overcome the shortcomings of existing single-function drugs or combination drug regimens.

[0008] This invention provides a recombinant protein that promotes burn repair. The recombinant protein consists of a human antimicrobial peptide LL-37 with a modified amino acid sequence and human epidermal growth factor (hEGF) with a modified amino acid sequence, linked by a linker. In the modified LL-37, the 26th amino acid position (Aspartic acid, Asp) is replaced with asparagine (Asn). In the modified hEGF, the 41st amino acid position (Arg) is replaced with lysine (Lys), and the 47th amino acid position (Leu) is replaced with valine (Val). Preferably, the linker is a (GGGGS)n flexible linker, where n is 1 or 2. More preferably, the complete amino acid sequence of the recombinant protein is shown in SEQ ID NO:3.

[0009] The present invention also provides a polynucleotide encoding the above-described recombinant protein. This polynucleotide has been optimized for efficient expression in hosts such as *E. coli*. A preferred polynucleotide is shown in SEQ ID NO:4.

[0010] The present invention further provides a recombinant expression vector containing the above-described nucleotide sequence, and a host cell containing the recombinant expression vector or whose genome has integrated the above-described nucleotide sequence. The host cell is preferably *Escherichia coli*.

[0011] The present invention also provides a pharmaceutical composition comprising the above-mentioned recombinant protein and a pharmaceutically acceptable carrier or excipient, suitable for preparation into various topical formulations, such as gels, ointments, creams, sprays or dressings.

[0012] The recombinant protein and pharmaceutical compositions comprising it involved in this invention can be used to prepare drugs for treating burns, promoting the repair of various acute and chronic wounds (such as diabetic foot ulcers, pressure ulcers, surgical incisions, etc.), or inhibiting bacterial infection of wounds. In a preferred embodiment, the drug is applied to the wound by topical administration.

[0013] The recombinant protein NV-Pro obtained in this invention combines the potent antibacterial activity of modified LL-37 with the highly efficient cell proliferation-promoting activity of modified hEGF. In vitro antibacterial experiments showed that the minimum inhibitory concentration (MIC) of NV-Pro against bacteria such as Escherichia coli was comparable to that of its parent antimicrobial peptide LL-37. Cellular experiments confirmed that NV-Pro significantly promoted the proliferation of human immortalized epidermal cells (HaCaT), with a proliferation-promoting effect consistent with hEGF. More importantly, in a rat full-thickness skin burn model, local application of NV-Pro significantly accelerated wound healing, with a therapeutic effect superior to using equivalent doses of LL-37 or hEGF alone. This indicates that the recombinant protein of this invention achieves synergistic and enhanced antibacterial and healing-promoting functions through a single molecule, providing an effective novel drug candidate molecule for solving the dual challenges of infection control and tissue regeneration in burn wound treatment.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Multifunctional integration, one drug with multiple effects: This invention creatively fuses the rationally modified antimicrobial peptide LL-37 with epidermal growth factor hEGF to obtain a single recombinant protein NV-Pro that simultaneously possesses efficient broad-spectrum antibacterial ability and strong ability to promote epidermal cell proliferation, thus achieving the organic unity of "anti-infection" and "promoting healing" functions.

[0015] (2) Synergistic effect and better therapeutic effect: In animal models, NV-Pro showed a better effect on promoting burn wound healing than LL-37 or hEGF alone.

[0016] (3) Reasonable molecular design and stable activity: By replacing amino acids at specific sites in LL-37 and hEGF (LL-37: D26N; hEGF: R41K, L47V), flexible linkers are used to connect them without affecting their spatial structure and function as much as possible, which minimizes the steric hindrance between functional domains and ensures that their biological activities can be expressed independently and fully.

[0017] (4) The preparation process is mature and easy to industrialize: The recombinant protein coding sequence provided by this invention has been codon optimized and can be expressed efficiently in the E. coli expression system. It can be purified by mature affinity chromatography process. The production process is simple and the cost is controllable, and it has good prospects for industrialization and clinical application.

[0018] (5) Simplify treatment plans and improve compliance: Integrating two treatment mechanisms into a single molecule is expected to reduce the complexity of clinical combination therapy, avoid potential interactions between different drugs, reduce treatment costs, and improve patients’ medication convenience and compliance. Attached Figure Description

[0019] Figure 1 This is an SDS-PAGE electrophoresis image of the recombinant protein NV-Pro after purification and enzyme digestion in Example 2, where M is the protein molecular weight standard and lane 1 is the NV-Pro protein sample in its non-reduced state.

[0020] Figure 2 The bar chart for the effects of various factors on HaCaT cell proliferation in Example 4 shows the relative cell proliferation rate of each experimental group relative to the control group.

[0021] Figure 3 This is a graph showing the change in wound healing rate over time in different treatment groups in a rat full-thickness skin burn model, as shown in Example 5. Detailed Implementation

[0022] 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.

[0023] Example 1: Sequence Design of Recombinant Proteins LL-37 is the only antimicrobial peptide in the cathelicidin family found in humans. It possesses an amphiphilic α-helix structure and exhibits inhibitory activity against both Gram-positive and Gram-negative bacteria, while also promoting angiogenesis and immunomodulation. The amino acid sequence of human LL-37 (SEQ ID No: 1: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was obtained from UniProt (https: / / www.uniprot.org / uniprotkb / P49913 / entry#sequences), and the amino acid Asp at position 26 was replaced with Asn. This increased the positive charge without altering the spatial structure, contributing to enhanced antimicrobial efficacy.

[0024] Human epidermal growth factor (hEGF) activates the tyrosine kinase signaling pathway by binding to the epidermal growth factor receptor, further promoting cell proliferation, tissue repair, and regeneration. The amino acid sequence Q6QBS2 (SEQ ID No: 2: NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR) was obtained from UniProt. Without altering the core functional region, it was rationally modified by replacing amino acid Arg at position 41 with Lys and amino acid Leu at position 47 with Val, to reduce its efficiency in being recognized and cleaved by serine proteases and matrix metalloproteinases.

[0025] A His tag and an enterokinase cleavage site were added to the N-terminus of the modified LL-37 sequence, and the modified hEGF was linked to the C-terminus using a G4S flexible linker to form the recombinant protein NV-Pro. The specific amino acid sequence is as follows: MGSSHHHHHHSSGDDDDKLLGDFFRKSKEKIGKEFKRIVQRIKNFLRNLVPRTESGGGGSGGGGSNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGEKCQYRDVKWWELR (SEQ ID No:3).

[0026] The mature recombinant protein sequence downstream of the EK restriction site was codon optimized using the online codon optimization tool ExpOptimizer. Escherichia coli was selected as the expression host, and BamHI and XhoI restriction sites were added at both ends. The optimized nucleotide sequence is GGATCCATGGGCAGCTCCCACCATCACCACCACCACAGCTCTGGCGATGACGACGATAAACTGCTGGGCGACTTCTTTCGCAAGTCCAAAGAGAAAATCGGCAAAGAGTTCAAACGTATCGTTCAGCGCATCAAAAACTTCCTGCGTAACCTGGTTCCGCGCACTGAGTCTGGTGGCGGTGGCAGCGGCGGTGGTGGCTCCAACTCTGATAGCGAGTGTCCGCTGAGCCATGACGGTTACTGTCTGCATGATGGCGTTTGCATGTACATCGAGGCCCTGGATAAATATGCGTGCAACTGTGTTGTAGGCTACATCGGCGAAAAATGCCAGTATCGTGACGTCAAGTGGTGGGAGCTGCGTTAACTCGAG (SEQ ID No:4).

[0027] Example 2: Expression and purification of NV-Pro in Escherichia coli Gene synthesis was performed by Suzhou Genewiz Technology Co., Ltd. The resulting plasmid containing the target gene was double-digested using BamHI-HF (NEB, Cat#R3136) and XhoI (NEB, Cat#R0146). The reaction system was as follows: 5 μL rCutSmart Buffer, 1 μg of plasmid containing the target gene, 1 μL BamHI-HF, and 1 μL XhoI were added sequentially to a 1.5 mL centrifuge tube, and the volume was brought to 50 μL with Nuclease-free Water. The 1.5 mL centrifuge tube was incubated at 37°C for 1 h, followed by enzyme inactivation at 65°C for 20 min. The linearized target gene fragment was recovered using a gel extraction kit (Promega, GP201A). Similarly, the pET28a+ vector was linearized using BamHI-HF and XhoI. Add 2 μL of T4 DNA Ligase Buffer (10×), 50 ng of linearized vector fragment, 50 ng of linearized target gene fragment, 1 μL of T4 DNA Ligase, and Nuclease-free Water to a PCR tube, bringing the total volume to 20 μL. Gently mix and incubate at room temperature for 2 h. Transform the ligation product into 100 μL of *E. coli* BL21 using the following steps: Place BL21 competent cells on ice until completely thawed, then add the ligation product, gently mix, incubate on ice for 30 min, incubate at 42°C for 90 s, incubate on ice for 3 min, add 1 mL of fresh antibiotic-free LB medium, and incubate at 37°C with a shaker at 220 rpm for 1 h. Centrifuge at 4000 rpm for 2 min, discard the supernatant, resuspend the cells in 100 μL of fresh antibiotic-free LB medium, and plate onto kanamycin-containing LB agar plates. Incubate overnight at 37°C with the plates inverted. The next day, pick single clones and have them sequenced by Suzhou Genewiz Technology Co., Ltd. Select single clones with correct sequences for cryopreservation.

[0028] BL21 glycerol bacteria containing the recombinant plasmid were streaked onto LB agar plates containing kanamycin and incubated overnight at 37°C with the plates inverted. The next day, a single plump positive colony was picked and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was then incubated at 37°C with shaking at 220 rpm for 12 h. The bacterial culture was then transferred to 500 mL of liquid medium at a 1:100 ratio and incubated at 37°C with shaking at 220 rpm until the bacterial growth rate reached OD. 600When the concentration of the bacterial culture reaches 0.6-0.8, add IPTG to a final concentration of 0.5 mM and incubate at 22°C and 180 rpm for 5 h. Centrifuge at 4°C and 8000 rpm for 10 min, discard the supernatant, and collect the bacterial cells. The bacterial cell pellet can be temporarily stored at -20°C. Prepare buffer A with the following composition: 20 mM Tris-HCl, 500 mM NaCl, pH=8.0. Resuspend the bacterial cells in buffer A at a ratio of 10 mL / g, mix thoroughly, and then sonicate. The sonication parameters are set as follows: power 200 W, sonication time 3 s, interval 5 s, total duration 30 min, temperature 4°C. Collect the sonicated bacterial culture, centrifuge at 4°C and 12000 rpm for 20 min, collect the supernatant, and discard the pellet.

[0029] Affinity chromatography was performed using a nickel column. First, equilibrate the column by adding 10 volumes of buffer A. Then, slowly add the supernatant from the disrupted bacterial culture to the column at a flow rate of approximately 1 mL / min. After loading, wash away most of the contaminating proteins with 10 column volumes of buffer B. Buffer B consists of 20 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole, pH 8.0. Elute the proteins using the following buffer: 20 mM Tris-HCl, 500 mM NaCl, and 250 mM imidazole, pH 8.0. Transfer the eluent to a dialysis bag, add buffer C, and dialyze overnight at 4°C, changing the buffer twice to completely remove imidazole. Buffer C consists of 20 mM Tris-HCl, 50 mM NaCl, and 2 mM CaCl2, pH 7.5. After determining the protein concentration, EK enzyme was added at a ratio of enzyme:protein = 1:500 (w / w), and digestion was performed at 25°C for 10 h. The digestion product was passed through a nickel column again, and the flow-through was collected. The flow-through was concentrated using a 3kD Amicon ultrafiltration tube, and the buffer was replaced with DPBS. 3 μg of purified NV-Pro protein was added to DTT-free loading buffer and analyzed by SDS-PAGE. The results are as follows. Figure 1 As shown in the figure, M is the protein molecular weight marker, lane 1 is the non-reduced protein with a molecular weight of about 11 kD, which is close to the theoretical value. The target band is clear and there are no impurities.

[0030] Example 3: Evaluation of the antibacterial activity of NV-Pro Escherichia coli (ATCC 25922) was streaked onto a fresh, antibiotic-free LB agar plate for activation. Single colonies were picked and inoculated into liquid LB medium. The culture was then incubated at 37°C with shaking at 220 rpm until the logarithmic growth phase. The bacterial culture was then diluted with medium to a final concentration of 2 x 10⁻⁶. 6CFU / mL. NV-Pro was serially diluted 2-fold using liquid LB medium, with the highest concentration being 400 μg / mL and the lowest concentration being 3.125 μg / mL. 50 μL of diluted bacterial culture was added to each well of a 96-well plate, and 50 μL of NV-Pro protein was added to each well of the experimental groups. The experimental groups and design are shown in Table 1. G1 was the positive control group, containing the same concentration of bacterial culture and wild-type LL-37 protein; G2 was the positive control group, containing the same concentration of bacterial culture and mutant LL-37 protein (D26N); G3 was the negative control group, containing the same concentration of bacterial culture and 50 μL / well of LB liquid medium; G4 was the blank control group, containing 100 μL / well of LB liquid medium; G6 was the negative control group, containing the same concentration of bacterial culture and 50 μL / well of hEGF protein; and G7 was the positive control group, containing mutant LL-37 protein and hEGF. After 12 h of incubation in a bacterial incubator, the OD values ​​were read using a microplate reader. 600 The lowest protein concentration at which the absorbance and OD values ​​showed no difference from the blank control group G3 was defined as the minimum inhibitory concentration (MIC). Experimental results showed that the MICs of G1 and G2 were consistent, and the two mutation sites in LL-37 did not affect its antibacterial activity. The MIC of NV-Pro was consistent with that of LL-37. WT The lack of difference indicates that the fusion-expressed NV-Pro still possesses the same antibacterial activity as LL-37.

[0031] Table 1. Experimental results of NV-Pro's anti-Escherichia coli activity evaluation Example 4: Evaluation of NV-Pro's Cell Proliferation-Promoting Effect Immortalized human epidermal cells (HaCaT) are core cells for reepithelialization in burn wounds and are suitable for evaluating the potential of drugs to promote burn repair. HaCaT cells in the logarithmic growth phase were digested and resuspended in culture medium to 5 × 10⁻⁶. 4 Cells / mL, 100 μL / well were seeded into 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. The culture medium was discarded, and 100 μL of medium containing different proteins was added to each well according to the group. The highest protein concentration was 200 μg / mL, and the remaining protein concentrations were serially diluted 10-fold starting from the highest concentration. Each group had 3 replicates. Group A was the control group with cells and blank medium, and Group G had no cells but an equal volume of blank medium. The experimental groupings are shown in Table 2 below. After 48 h, 10 μL of CCK-8 solution was added to each well, and the plates were incubated in the dark for 1 h. OD was measured using a microplate reader. 450 Value. Relative cell proliferation rate = (OD value of experimental group - OD value of group G) / (OD value of group A - OD value of group G) × 100%, and the experimental results are expressed as follows: Figure 2LL-37 alone cannot promote the proliferation of HaCaT. Compared with hEGF, NV-Pro has a consistent cell proliferation curve, which means that it retains the function of hEGF.

[0032] Table 2. Experimental Groups Example 5: Evaluation of the therapeutic effect of NV-Pro in a rat model of full-thickness skin burns Eight-week-old SPF-grade male SD rats were selected and randomly divided into groups after 5 days of acclimatization. All rats underwent modeling, and were fasted for 12 hours before surgery, but water was allowed. After isoflurane anesthesia, the fur on the back of the rats was shaved, disinfected with povidone-iodine, and then deiodinated with 75% alcohol. A 10mm diameter circular poultice was used to apply 90℃ hot water to the back skin for 10 seconds to create a wound with full-thickness epidermis and part of dermis damage. The wounds were allowed to air dry naturally after surgery, without additional debridement, and the rats were housed individually to prevent biting. Four hours after surgery, appropriate drug treatment was initiated according to the group assignments, with the drug applied to the wound twice daily for 14 consecutive days. The length (a) and width (b) of the wound were measured with calipers every other day, and the wound area was recorded. The wound area = πab / 4, and the wound healing rate = (initial wound area - current wound area) / initial wound area * 100%. Figure 3 It is known that, at the same dosage, NV-Pro has a faster wound healing speed than human epidermal growth factor or LL-37 alone, which helps repair full-layer dermal damage and has good drug development potential.

[0033] Table 3. Experimental Groups Eight hours after the last administration, the animal was euthanized, and the full-thickness skin from the wound was harvested, weighed, and cut into 35mm pieces. Physiological saline was added at a concentration of 5 mL / g, and the mixture was homogenized using a bead mill. The homogenate was centrifuged at 12000 rpm / min for 15 min at 4°C. The supernatant was transferred to a new 1.5 mL EP tube, and a small amount of the supernatant was used to determine the total protein concentration using BCA. An equal volume of supernatant (containing 50 μg of total protein) was taken, and 50 mM NH4HCO3 was added to a total volume of 100 μL. RapiGestSF was added to a final concentration of 0.05%, and the mixture was incubated at 56°C for 30 min. 100 mM DTT was added to a final concentration of 10 mM, and the mixture was incubated at 56°C for 30 min to open the disulfide. The mixture was cooled to room temperature, and 200 mM IAA was added to a final concentration of 20 mM in the dark. The mixture was reacted at room temperature in the dark for 30 min. 10× TPCK was added to treat trypsin, and the mixture was incubated overnight at 37°C with low-speed shaking. Add formic acid to a final concentration of 1%, mix well, let stand at room temperature for 10 min, centrifuge at 12000 rpm for 10 min, and collect the supernatant. Identify the drug concentration in the wound using LC-MS / MS. LL-37 WT / LL-37mut The LL-37 terminus of NV-Pro is the FKRIVQRIK peptide, with a precursor ion of 569.3 and daughter ions of 840.5 and 614.4. The EGFR terminus of hEGFR and NV-Pro is the YIEALDK peptide, with a precursor ion of 405.7 and daughter ions of 620.3 and 491.3. The quantitative results are shown in Table 4. NV-Pro exhibits better residual levels of LL-37 and hEGFR peptides 8 hours after administration, and its elimination from the affected area is more slow, effectively compensating for the short half-life issue associated with monotherapy.

[0034] Table 4. Determination of residual wound medication 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 protein that promotes burn repair, characterized in that, It includes a human antimicrobial peptide LL-37 with modified amino acid sequence and a human epidermal growth factor hEGF with modified amino acid sequence, wherein the modified LL-37 and the modified hEGF are linked by a linker. The modified LL-37 amino acid sequence is based on the sequence shown in SEQ ID NO:1, with the aspartic acid Asp at position 26 replaced by asparagine Asn. The modified hEGF amino acid sequence is based on the sequence shown in SEQ ID NO:2, with arginine (Arg) at position 41 replaced by lysine (Lys) and leucine (Leu) at position 47 replaced by valine (Val). The connector is a (GGGGS)n flexible connector, where n is an integer ≥ 1.

2. The recombinant protein according to claim 1, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO:

3.

3. A polynucleotide encoding the recombinant protein of any one of claims 1-2.

4. A recombinant expression vector, characterized in that, It comprises the polynucleotide as described in claim 3.

5. A host cell, characterized in that, It includes the recombinant expression vector of claim 4, or the polynucleotide of claim 3 is integrated into the genome.

6. A pharmaceutical composition, characterized in that, It comprises the recombinant protein as described in any one of claims 1-2 and a pharmaceutically acceptable carrier.

7. The use of the recombinant protein according to any one of claims 1-2 or the pharmaceutical composition according to claim 6 in the preparation of a medicament for treating burns, promoting wound healing, or inhibiting bacterial infection of wounds.

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