利用大肠杆菌合成抗氧化蛋白材料及其在促进创面愈合中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2026-0087

摘要/Abstract

摘要：

目的构建能够自组装成水凝胶的抗氧化蛋白材料，探究其促进细胞氧化损伤修复和伤口愈合的作用。方法构建重组质粒，将已报道的自组装弹性蛋白骨架，与具有抗氧化活性的氨基酸序列进行融合，导入大肠杆菌发酵并纯化。通过自由基清除实验筛选出性能最优的蛋白，用细胞氧化损伤修复实验和大鼠创面愈合实验探究其应用价值。结果经过大肠杆菌表达并纯化的融合蛋白在1%浓度下可自组装形成水凝胶。融合抗氧化氨基酸序列后，蛋白自由基清除能力显著提升。其中80-2-MPH对DPPH·、ABTS⁺·和·OH的清除率分别达41.1%、66.8%和57.2%，较原始蛋白80-2-RGD（清除率分别为20.4%、50.0%和21.8%）显著提高（P<0.000 1）。80-2-MPH生物相容性良好，1 g/L浓度时相对细胞活性达117.6%。在细胞氧化损伤修复实验中，80-2-MPH修复组细胞活性为49.6%，显著优于空白组（28.0%）及谷胱甘肽（36.7%）、维生素C（40.9%）修复组；80-2-MPH修复组细胞内活性氧（reactive oxygen species, ROS）含量与谷胱甘肽组无显著差异。大鼠全层创面愈合实验中，在第10天，80-2-MPH水凝胶修复组伤口闭合率达93.6%，显著高于空白组（74.8%）和商用水凝胶敷料组（84.5%）。组织染色结果显示，80-2-MPH水凝胶可有效促进胶原沉积与真皮层修复。结论通过大肠杆菌表达成功获得了能够自组装成水凝胶且具有抗氧化能力的蛋白材料80-2-MPH。该蛋白能够降低氧化应激细胞的ROS含量、促进细胞的氧化损伤修复。其自组装形成的抗氧化水凝胶敷料能够有效促进大鼠创面愈合。

关键词: 重组蛋白, 功能蛋白材料, 蛋白质工程, 蛋白纯化, 抗氧化, 水凝胶, 氧化损伤修复

Abstract:

Objective This study aimed to construct a self-assembling antioxidant protein material and to evaluate its effects on repairing oxidative cellular damage and promoting wound healing. Method We constructed recombinant plasmids by fusing a previously reported self-assembling elastin-like polypeptide scaffold with antioxidant amino acid sequences. We then transformed the constructs into Escherichia coli for fermentation and purification. We selected the protein with the optimal performance through free radical scavenging assays and further evaluated its application potential using cellular oxidative damage repair assays and rat wound healing experiments. Result The recombinant protein expressed and purified from E. coli self-assembled into a hydrogel at a concentration of 1%. After fusion with the antioxidant amino acid sequence, the free radical scavenging capacity of the protein was significantly enhanced. The scavenging rates of 80-2-MPH for 2,2-diphenyl-1-picrylhydrazyl (DPPH·), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺·), and hydroxyl radical (·OH) reached 41.1%, 66.8%, and 57.2%, respectively, which were significantly higher than those of the original protein 80-2-RGD (20.4%, 50.0%, and 21.8%, respectively; P<0.000 1). The 80-2-MPH protein exhibited good biocompatibility, with a relative cell activity of 117.6% at a concentration of 1 g/L. In the cellular oxidative damage repair experiment, the cell activity in the 80-2-MPH repair group was 49.6%, significantly higher than that of the control group (28.0%), as well as the glutathione (36.7%) and vitamin C (40.9%) treatment groups. The intracellular reactive oxygen species (ROS) content in the 80-2-MPH group showed no significant difference compared with the glutathione group. In the rat full-thickness wound healing experiment, the 80-2-MPH hydrogel group showed a wound closure rate of 93.6% on day 10, which was significantly higher than that of the control group (74.8%) and the commercial hydrogel dressing group (84.5%). Histological staining results showed that the 80-2-MPH hydrogel effectively promoted collagen deposition and dermal repair. Conclusion An antioxidant protein material, 80-2-MPH, capable of self-assembling into a hydrogel, was successfully expressed in E. coli. This protein reduced intracellular ROS levels in oxidatively stressed cells and promoted the repair of oxidative damage. The hydrogel dressing formed by the self-assembly of this protein effectively accelerates wound healing in rats.

Key words: recombinant protein, functional protein materials, protein engineering, protein purification, antioxidant, hydrogel, oxidative damage repair

赵梦蝶, 薛卫士, 王家棒, 李凌, 杨如梦, 吴俊俊. 利用大肠杆菌合成抗氧化蛋白材料及其在促进创面愈合中的应用[J]. 生物技术通报, 2026, 42(9): 342-354.

ZHAO Meng-die, XUE Wei-shi, WANG Jia-bang, LI Ling, YANG Ru-meng, WU Jun-jun. Synthesis of Antioxidant Protein Material by Escherichia coli and Its Applications in Promoting Wound Healing[J]. Biotechnology Bulletin, 2026, 42(9): 342-354.

图/表 9

表1 蛋白外露活性位点的氨基酸序列及对应碱基序列和实验中所用引物序列

Table 1 Amino acid and corresponding nucleotide sequences of the exposed active site of the protein, and primer sequences used in this study

氨基酸序列 AA sequences	名称 Name	碱基序列 Base sequences	引物序列 Primer sequences （5′-3′）	参考文献 References
AMRLTYNKPCLYGT	RP1	GCTATGAGGCTAACATATAATAAACCCTGTCTGTACGGTACC	F1： GCTATGAGGCTAACATATAATTGACTCGAGCACCACCACCA R1： ATTATATGTTAGCCTCATAGCACCGCTACCACCGCTACCCGGGGCGC F2： AAACCCTGTCTGTACGGTACCTGACTCGAGCACCACCACC R2：GGTACCGTACAGACAGGGTTTATTATATGTTAGCCTCATAGCACCGCTAC	［21］
LMGQW	MPH	CTGATGGGCCAGTGGTAA	F： CTGATGGGCCAGTGGTAATGACTCGAGCACCACCACC R： TTACCACTGGCCCATCAGACCGCTACCACCGCTACCCGGC	［22］
SDITRPGGNM	PP	AGCGATATTACCCGCCCGGGCGGCAACATGTAA	F：AGCGATATTACCCGCCCGGGCGGCAACATGTAATGACTCGAGCACCACCACC R：TTACATGTTGCCGCCCGGGCGGGTAATATCGCTACCGCTACCACCGCTACCCGGC	［23］
YYIVS	Ⅳ5C	TACTACATCGTGAGCTAA	F： TACTACATCGTGAGCTAATGACTCGAGCACCACCACC R：TTAGCTCACGATGTAGTAACCGCTACCACCGCTACCCGGC	［24］
SETYELRK	ABA	AGCGAAACCTATGAACTGCGCAAATAA	F： AGCGAAACCTATGAACTGCGCAAATAATGACTCGAGCACCACCACC R： TTATTTGCGCAGTTCATAGGTTTCGCTACCGCTACCACCGCTACCCGGC	［25］
GRGDSPG	RGD	GGCCGCGGCGATAGCCCGGGC	/	［19］

图1 大鼠全层创面愈合实验的实验流程图

Fig. 1 Flowchart of the rat full-thickness wound healing experiment

图2 基于自组装弹性蛋白序列构建抗氧化蛋白A：通过反向PCR构建重组蛋白表达质粒方法示意图。B：单个蛋白分子的一级结构以及自组装形成水凝胶结构的示意图。C：AlphaFold 3模拟自组装后蛋白的结构图，黄色部分为其中一个蛋白分子的自组装序列，橙色部分为弹性蛋白序列，红色部分为外露的活性位点

Fig. 2 Construction of antioxidant proteins based on self-assembling elastin-like sequencesA: Schematic diagram of recombinant protein expression plasmid construction via inverse PCR. B: Schematic diagram of the primary structure of a single protein molecule and the self-assembled hydrogel structure. C: Structure diagram of the self-assembled protein predicted by AlphaFold 3. The yellow region represents the self-assembling sequence of one protein molecule, the orange region represents the elastin-like sequence, and the red region represents the exposed active site

图3 蛋白表达与纯化的蛋白胶图A：蛋白发酵时，诱导大肠杆菌表达前后蛋白胶图。M：Marker；1：诱导前；2：诱导后。 B-E：分别为80-2-MPH、80-2-PP、80-2-RP1、80-2-Ⅳ5C、80-2-ABA蛋白纯化过程的蛋白胶图，分子量均为27 kD左右。M：Marker；1：细胞破碎液；2：流穿液；3：洗杂后镍柱洗脱蛋白；4：脱盐后蛋白

Fig. 3 Protein expression and purification gel imagesA: Gel images of protein expression in E. coli before and after induction during protein fermentation. M: Marker; 1: before induction; 2: after induction. B-E: Gel images showing the purification of 80-2-MPH, 80-2-PP, 80-2-RP1, 80-2-Ⅳ5C, and 80-2-ABA, respectively. The molecular weight of each protein was approximately 27 kD. M: Marker; 1: cell lysate; 2: flow-through; 3: Ni-column eluted protein after washing; 4: desalted protein

图4 化学实验表征不同蛋白的抗氧化性质A：不同蛋白DPPH自由基清除率。B：ABTS自由基清除率。C：不同蛋白·OH清除率。D：不同浓度80-2-MPH的自由基清除能力（照片为自由基清除效果照片）。****P<0.000 1，数据均表示为平均值±标准差（n=3），下同

Fig. 4 Chemical experiment characterizes the antioxidant properties of different proteinsA: DPPH· scavenging rate of different proteins. B: ABTS+· free radical scavenging rate. C: ·OH scavenging rate of different proteins. D: Free radical scavenging ability of different concentrations of 80-2-MPH. The photos show the effect of free radical scavenging. ****P<0.000 1, data are reported as mean ± SD (n=3), the same below

图5 水凝胶成胶性质的表征A：不同蛋白按1%（w/v）溶解于生理盐水，未成胶。从左到右分别为80-2-Ⅳ5C，80-2-PP，80-2-RP1，80-2-ABA和80-2-MPH。B：蛋白静置后自组装成水凝胶。C：浓度为4%（w/v）蛋白成胶后用注射器添加至猪皮缺损处，模拟蛋白作为伤口敷料应用时的场景。D：扫描电子显微镜观察5%（w/v）浓度水凝胶的网络结构。E：扫描电子显微镜观察10%（w/v）浓度水凝胶的网络结构

Fig. 5 Characterization of hydrogel gelation propertiesA: Different proteins were dissolved in physiological saline at a concentration of 1% (w/v), and no gelation occurred. From left to right: 80-2-Ⅳ5C, 80-2-PP, 80-2-RP1, 80-2-ABA, and 80-2-MPH. B: The protein self-assembled into a hydrogel after standing. C: After gelation at 4% (w/v), the protein hydrogel was applied to a porcine skin defect using a syringe to mimic its use as a wound dressing. D: Scanning electron microscopy images showing the network structure of the hydrogel at 5% (w/v). E: Scanning electron microscopy images showing the network structure of the hydrogel at 10% (w/v)

图6 80-2-MPH蛋白的生物活性A：80-2-MPH的生物相容性验证。含有不同浓度80-2-MPH的完全培养基培养细胞，CCK-8试剂盒检测细胞活力。B：显微镜观察各组细胞形态。***P<0.001

Fig. 6 Biological activity of 80-2-MPH proteinA: Verification of the biocompatibility of 80-2-MPH. Cells were cultured in complete medium containing different concentrations of 80-2-MPH, and cell viability was detected using the CCK-8 kit. B: Scanning electron microscopy images showing cell morphology in each group. ***P<0.001

图7 80-2-MPH对细胞氧化损伤的修复作用以及对细胞中活性氧的清除作用A：损伤后修复12 h和24 h的细胞形态。 B：修复12 h时细胞活死染。C：DCFH-DA检测细胞中的活性氧。D：修复12 h时CCK-8测定各组的细胞活性。数据均表示为平均值±标准差（n=4）。E：流式细胞仪检测细胞中的相对活性氧含量。*P<0.05

Fig. 7 Effect of 80-2-MPH on the repair of cellular oxidative damage and the scavenging of intracellular reactive oxygen speciesA: Cell morphology at 12 h and 24 h after damage repair. B: Live/dead cell staining at 12 h after damage repair. C: Detection of reactive oxygen species in cells using DCFH-DA. D: Cell viability was measured by CCK-8 assay in each group at 12 h of repair. Data are reported as means ± SD (n=4). E: Detection of relative reactive oxygen species content in cells using flow cytometry. *P<0.05

图8 大鼠全层创面闭合情况A：定时记录不同组大鼠的伤口愈合情况。B：不同组大鼠伤口闭合率计算。数据均表示为平均值±标准差（n=6）。C：皮肤样品的H&E染色和Masson染色

Fig. 8 Closure of full-thickness wounds in ratsA: Wound healing in different groups of rats was recorded at regular intervals. B: Wound closure rate in different groups of rats. Data are reported as means ± SD (n=6). C: H&E staining and Masson staining of skin samples

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