Recombinant protein-based hydrogel, preparation method and application thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a hydrogel based on recombinant proteins, a preparation method and application. The application uses a recombinant protein expression system to scale up preparation of protein elements, realizes high mechanical strength of a hydrogel structure, ROS triggered release and cell targeting function through a SpyTag / SpyCatcher and Coil-Coil double crosslinking mechanism and ULD tetramer self-assembly, and the preparation process is simple and suitable for precise drug delivery and tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a hydrogel based on recombinant proteins, its preparation method, and its application. Background Technology

[0002] Hydrogels are three-dimensional hydrophilic network structures formed by physical or chemical cross-linking of natural or synthetic polymers. They can absorb large amounts of water or biological fluids and maintain a semi-solid form. Due to their excellent biocompatibility, biodegradability, mechanical stability / protein stability, flexibility, and the ability to control the release of various therapeutic agents in space and time, they have shown significant application value in drug delivery, regenerative medicine, and other fields. Traditional hydrogels are mainly constructed based on synthetic polymers (such as polyethylene glycol PEG, hyaluronic acid-methylcellulose, etc.) or naturally extracted biomacromolecules (such as DNA, peptides, and proteins), but their applications face the following bottlenecks: (1) The material properties (such as mechanical strength and degradation rate) of natural protein hydrogels extracted from biological tissues are difficult to control precisely, and there are batch-to-batch differences and potential immunogenicity issues. (2) Synthetic polymer hydrogels lack bioactive sites and are difficult to simulate the dynamic microenvironment of natural tissues.

[0003] Currently, recombinant protein-based hydrogels are attracting attention because they retain the high biocompatibility and bioabsorbable components similar to natural protein gels, while offering greater self-defined tunability without the need for synthetic chemistry integration. However, existing protein hydrogels mostly rely on random cross-linking, resulting in unclear network structures, ambiguous cross-linking mechanisms, insufficient mechanical properties, and limited functional expansion. Although the SpyTag / SpyCatcher system and Coil-Coil interactions have been used for cross-linking, single mechanisms are insufficient to simultaneously ensure stability and dynamic responsiveness. Furthermore, traditional materials lack intelligent responsiveness to pathological microenvironments (such as ROS). Summary of the Invention

[0004] To address the needs of existing technologies, the purpose of this invention is to provide a recombinant protein-based hydrogel, its preparation method, and its applications. This invention utilizes a multi-mechanism synergistic strategy integrating SpyTag / SpyCatcher covalent crosslinking, Coil-Coil nonvalent assembly, and ULD tetramer self-assembly to achieve, for the first time, a programmable protein hydrogel possessing high mechanical strength, dual ROS / pH responsiveness, and RGD targeting function. Its preparation process is simple and suitable for precise drug delivery and tissue repair.

[0005] Specifically, the present invention provides the following technical solution: A first aspect of the present invention provides a hydrogel based on recombinant proteins, formed by two fusion proteins through a covalent and non-covalent double crosslinked network: ULD-SpyCatcher fusion protein (ULDC), wherein the ULD protein (ubiquitin-like domain protein) is a cysteine-rich tetrameric protein that is linked to SpyCatcher through an elastin-like protein (ELP). Its N-terminus also contains an RGD sequence. The amino acid sequence is shown in SEQ ID NO:1 and the nucleotide sequence is shown in SEQ ID NO:2. SpyTag-Coil-SpyTag fusion protein (TCT), wherein the Coil protein (helical domain protein) is connected to the SpyTag sequences on both sides through an elastin-like protein (ELP), the amino acid sequence is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4; The hydrogel forms a double crosslinked network through SpyTag / SpyCatcher covalent crosslinking, Coil-Coil nonvalent interaction, and ULD tetramer self-assembly.

[0006] Preferably, the molar mixing ratio of the ULD-SpyCatcher fusion protein and the SpyTag-Coil-SpyTag fusion protein is 1:2.

[0007] Preferably, the total protein concentration of the hydrogel is 8 wt%.

[0008] Preferably, the hydrogel has a pore size of less than 50 micrometers and a porous structure.

[0009] Preferably, the surface structure of the hydrogel disintegrates within 5 minutes under 1 mM H2O2 conditions, exhibiting reactive oxygen species (ROS) responsiveness.

[0010] Preferably, the hydrogel is stable in a neutral pH (PBS, pH 7.5) and slowly degrades in an acidic (SGF, pH 1.0) or alkaline (SIF, pH 8.5) environment.

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned recombinant protein-based hydrogel, comprising the following steps: Expression vectors were constructed, transformed into host cells and induced to express. The ULD-SpyCatcher and SpyTag-Coil-SpyTag fusion proteins were then purified by Ni-NTA affinity chromatography. The purified ULD-SpyCatcher and SpyTag-Coil-SpyTag fusion proteins were then dialyzed and freeze-dried, and then mixed in phosphate-buffered saline (PBS) for a water bath reaction to obtain the final product.

[0012] Preferably, the expression vector is pET32a, and the host cell includes Escherichia coli. More preferably, the host cell is Escherichia coli BL21(DE3).

[0013] Preferably, the conditions for inducing expression are as follows: when the OD600 reaches 0.6~0.8, add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM, and induce expression at 16°C for 16-20 hours.

[0014] Preferably, the elution buffer for the Ni-NTA affinity chromatography is a 50 mM HEPES buffer (pH 7.5) containing 50 mM NaCl.

[0015] Preferably, the dialysis uses deionized water and the dialysis time is 20-30 hours.

[0016] Preferably, the water bath reaction is carried out at a temperature of 37°C for 1 hour.

[0017] A third aspect of the present invention provides the application of the recombinant protein-based hydrogel described in the first aspect in drug delivery or tissue engineering.

[0018] The beneficial effects achieved by one or more of the above technical solutions of the present invention are as follows: (1) This invention utilizes a recombinant protein expression system (such as Escherichia coli BL21(DE3)) to prepare protein elements (ULD-SpyCatcher and SpyTag-Coil-SpyTag fusion proteins) on a large scale, avoiding complex synthetic chemical modifications; specifically, hydrogels are formed through a mild and efficient cross-linking reaction (SpyTag / SpyCatcher covalent cross-linking, Coil-Coil non-covalent interaction and ULD tetramer self-assembly), the process is controllable, has good reproducibility, and is suitable for further functionalization.

[0019] (2) The hydrogel prepared by the present invention has reactive oxygen species (ROS) responsiveness: the surface rapidly disintegrates within 5 minutes under 1 mM H2O2 conditions, which is suitable for intelligent drug release in pathological microenvironments (such as inflammation or tumor sites) and can be used to develop intelligent responsive gel carriers. It exhibits pH responsiveness: it is stable in a neutral environment (pH 7.5) and degrades slowly in acidic (pH 1.0) or alkaline (pH 8.5) environments, thus adapting to the needs of different physiological or pathological conditions.

[0020] (3) The hydrogel prepared by this invention has targeting function and biocompatibility: RGD sequence targeting: The N-terminus of the ULD-SpyCatcher fusion protein contains an RGD sequence, which endows the hydrogel with specific adhesion to cells with high integrin expression (such as tumor cells) and enhances the targeted delivery effect. Low cytotoxicity: In vitro experiments showed that the hydrogel had no significant toxicity to MC38 cells in the concentration range of 0-100 μM, demonstrating good biocompatibility.

[0021] (4) The hydrogel prepared by the present invention maintains the macroscopic gel structure after being stored at room temperature in the dark for 12 days, and has good stability. The porous structure (pore size <50 micrometers) is conducive to cell migration, nutrient exchange and drug loading and release.

[0022] (5) This invention provides a recombinant protein hydrogel with good mechanical properties, laying the material foundation for constructing drug delivery systems and tissue engineering materials. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 This is the plasmid map of pET32a-ULD-SpyCatcher in Example 1 of the present invention; Figure 2 This is the plasmid map of pET32a-SpyTag-Coil-SpyTag in Example 1 of the present invention; Figure 3 This is an SDS-PAGE image of the pET32a-SpyTag-Coil-SpyTag and pET32a-ULD-SpyCatcher proteins expressed and purified in Example 1 of this invention. Figure 4 This document describes the preparation and characterization of SpyTag-Coil-SpyTag and ULD-SpyCatcher hydrogels in Example 1 of this invention, where A is a schematic diagram of the hydrogels; B is an SDS-PAGE image of the scaffold protein crosslinking; and C is a SEM characterization of the hydrogels. Figure 5 These are macroscopic images of different types of fusion proteins after the reaction, where a is a macroscopic image of the fusion protein constructed by SpyTag-SpyTag and ULD-SpyCatcher after the reaction, and b is a macroscopic image of the fusion protein hydrogel constructed by SpyTag-Coil-SpyTag and ULD-SpyCatcher. Figure 6 Stability analysis of the hydrogel prepared in Example 1 of this invention; Figure 7 Analysis of the response and release characteristics of the hydrogel prepared in Example 1 of this invention; Figure 8 Rheological analysis of the hydrogel prepared in Example 1 of this invention; Figure 9 The in vitro toxicity test of the hydrogel prepared in Example 1 of this invention; Figure 10 This shows the in vitro adhesion of the hydrogel prepared in Example 1 of the present invention. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] As mentioned above, hydrogels are widely used in the biomedical field due to their high biocompatibility and tunable drug release capabilities. However, most existing protein hydrogel construction strategies rely on random chain extension and cross-linking reactions between linear protein polymers, resulting in unclear network structures, ambiguous cross-linking mechanisms, and limitations on the mechanical properties and functional tunability of the materials. Therefore, this invention provides a recombinant protein hydrogel that achieves high mechanical strength, ROS-triggered release, and cell-targeting functions through a SpyTag / SpyCatcher and Coil-Coil dual cross-linking mechanism and ULD tetramer self-assembly.

[0027] Specifically, this invention designs a scaffold protein based on tetrameric protein and SpyTag / SpyCatcher. One scaffold has a Coil in the middle of the chain and a SpyTag(T) at both the N- and C-termini, namely SpyTag-ELP-Coil-ELP-SpyTag (TCT, 41.1 kDa). The other scaffold uses ULD-ELP-Spycatcher (ULDC, 50.5 kDa) recombinant protein as a chemical cross-linking agent. The N-terminus of ULD also contains an RGD sequence to increase the adhesion and targeting ability of the hydrogel.

[0028] A first typical embodiment of the present invention provides a hydrogel based on recombinant proteins, formed by two fusion proteins through a covalent and non-covalent double crosslinked network: ULD-SpyCatcher fusion protein (ULDC), wherein the ULD protein is a cysteine-rich tetrameric protein that is linked to SpyCatcher via an elastin-like protein (ELP), and its N-terminus also contains an RGD sequence. The amino acid sequence is shown in SEQ ID NO:1; the nucleotide sequence is shown in SEQ ID NO:2. SpyTag-Coil-SpyTag fusion protein (TCT), wherein the Coil protein is linked to the SpyTag sequences on both sides via an elastin-like protein (ELP), the amino acid sequence of which is shown in SEQ ID NO:3; and the nucleotide sequence of which is shown in SEQ ID NO:4; The hydrogel forms a double crosslinked network through SpyTag / SpyCatcher covalent crosslinking, Coil-Coil nonvalent interaction, and ULD tetramer self-assembly.

[0029] In one or more embodiments of this implementation, the molar mixing ratio of the ULD-SpyCatcher fusion protein and the SpyTag-Coil-SpyTag fusion protein is 1:2.

[0030] In one or more embodiments of this implementation, the total protein concentration of the hydrogel is 8 wt%.

[0031] In one or more embodiments of this implementation, the hydrogel has a pore size of less than 50 micrometers and has a porous structure.

[0032] In one or more embodiments of this implementation, the surface structure of the hydrogel disintegrates within 5 minutes under 1 mM H2O2 conditions, exhibiting reactive oxygen species (ROS) responsiveness.

[0033] In one or more embodiments of this implementation, the hydrogel is stable in a neutral pH (PBS, pH 7.5) and slowly degrades in an acidic (SGF, pH 1.0) or alkaline (SIF, pH 8.5) environment.

[0034] A second typical embodiment of the present invention provides a method for preparing the above-mentioned recombinant protein-based hydrogel, comprising the following steps: Expression vectors were constructed, transformed into host cells and induced to express. The ULD-SpyCatcher and SpyTag-Coil-SpyTag fusion proteins were then purified by Ni-NTA affinity chromatography. The purified ULD-SpyCatcher and SpyTag-Coil-SpyTag fusion proteins were then dialyzed and freeze-dried, and then mixed in phosphate-buffered saline (PBS) for a water bath reaction to obtain the final product.

[0035] In one or more embodiments of this implementation, the expression vector is pET32a.

[0036] In one or more embodiments of this implementation, the host cell includes Escherichia coli, and more specifically, the host cell is Escherichia coli BL21(DE3).

[0037] In one or more embodiments of this implementation, the conditions for inducing expression are as follows: when the OD600 reaches 0.6~0.8, add isopropyl-β-D-thiogalactoside at a final concentration of 0.1 mM, and induce expression at 16°C for 16-20 hours.

[0038] In one or more embodiments of this implementation, the C-terminus of the expression vector contains a 6XHis tag encoding gene.

[0039] Preferably, the elution buffer for the Ni-NTA affinity chromatography is a 50 mM HEPES buffer (pH 7.5) containing 50 mM NaCl.

[0040] Preferably, the dialysis uses deionized water and the dialysis time is 20-30 hours.

[0041] Preferably, the water bath reaction is carried out at a temperature of 37°C for 1 hour.

[0042] A third typical embodiment of the present invention provides an application of the above-described recombinant protein-based hydrogel in drug delivery or tissue engineering.

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] Example 1: This example provides a hydrogel based on recombinant protein and its preparation method. (1) Preparation of scaffold proteins: In order to obtain recombinant TCT and ULDC proteins, pET32a-ULD-SpyCatcher and pET32a-SpyTag-Coil-SpyTag expression vectors were constructed (e.g., Figures 1-2(As shown), the nucleotide sequences are SEQ ID NO:5 and SEQ ID NO:6. The recombinant expression vector was transformed into *E. coli* BL21(DE3), yielding several strains. Different strains were then cultured in LB medium at 37°C. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM for induction at 16°C. After approximately 20 h, all cells were centrifuged at 8000 rpm for 10 min. The cell particles were resuspended in 50 mM HEPES buffer (pH 7.5) containing 50 mM NaCl. Cells were lysed by sonication, and the supernatant was collected after centrifugation. Since all proteins used in this study were His-labeled, the supernatant was purified using nickel-filled resin, followed by dialysis with distilled water and freeze-drying. Figure 3 As shown, the expressed and purified proteins were analyzed by 12.5% ​​SDS-PAGE, and both purified proteins showed a single band.

[0045] (2) Preparation and characterization of hydrogels The purified protein from step (1) was dialyzed, freeze-dried, and then dissolved in phosphate buffer (1×PBS) to prepare an 8 wt% solution (e.g. Figure 4 As shown in Figure A), TCT and ULDC are then mixed at a molar ratio of 2:1. A covalent reaction occurs immediately upon mixing of the scaffold proteins, forming different multimers (such as dimers, trimers, and tetramers). The formation of multimeric scaffold proteins is rapid (e.g., ...). Figure 4 As shown in Figure B), and as time goes on, it gradually forms a colloidal state, which is the result.

[0046] (3) After the hydrogel was prepared in step (2), it was frozen overnight at -80°C. The frozen protein hydrogel was cut crosswise with a knife to observe the cross-section. The hydrogel was then freeze-dried in a freeze dryer. The freeze-dried protein hydrogel was placed on a stage for a scanning electron microscope with the observation side facing upwards. The freeze-dried hydrogel was coated with a layer of platinum under vacuum conditions. The structure of the sample was then observed and photographed. The internal structure of the freeze-dried hydrogel was analyzed by SEM. The results are as follows: Figure 4 As shown in Figure C, the hydrogel has a porous structure, the surface of the protein is smooth, and the pore size is less than 50 micrometers.

[0047] Comparative Example 1: To compare the performance of the fusion protein hydrogel constructed in Example 1, two sets of protein scaffolds were designed in this comparative example: a) SpyTag-SpyTag (TT) and ULD-SpyCatcher (ULDC); b) SpyTag-Coil-SpyTag (TCT) and ULD-SpyCatcher (ULDC). Based on this, expression vectors pET32a-SpyTag-SpyTag (nucleotide sequence SEQ ID NO:7), pET32a-SpyTag-Coil-SpyTag, and pET32a-ULD-SpyCatcher were further constructed. The recombinant expression vectors were transformed into Escherichia coli BL21(DE3), and several strains were obtained. Different strains were cultured in LB medium at 37°C. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 0.1 mM for induction at 16°C for approximately 20 h. Afterward, all cells were centrifuged at 8000 rpm for 10 min. Cell particles were resuspended in 50 mM HEPES buffer (pH 7.5) containing 50 mM NaCl. Cells were lysed by sonication. The supernatant was collected after centrifugation. Since all proteins used in this study were His-labeled, the supernatant was purified with nickel-filled resin. The proteins were then dialyzed with distilled water and freeze-dried to obtain protein powder.

[0048] The purified protein powder was dissolved in phosphate-buffered saline (1×PBS) to prepare an 8 wt% solution. The TT / TCT and ULDC solutions were mixed separately at a molar ratio of 2:1, and the results are as follows: Figure 5 As shown, no true hydrogel was formed after the reaction of TT and ULDC. Figure 5 As shown in Figure a), TCT and ULDC react to form a gel that does not flow when inverted (as shown in Figure a). Figure 5 (As shown in b). Therefore, in subsequent experiments, the fusion protein scaffolds ULD-SpyCatcher and SpyTag-Coil-SpyTag were selected for relevant performance tests.

[0049] Example 2: This experiment analyzes the stability of the hydrogel prepared in Example 1. The hydrogel stability test consisted of two parts: one part involved determining the storage time of the hydrogel. The hydrogel was placed in a 2 mL glass bottle and then stored at room temperature in the dark. The hydrogel state was observed at specific time points (1 day, 2 days, 4 days, 8 days, and 12 days). The results are as follows: Figure 6 As shown, the macroscopic gel structure of the hydrogel remained unchanged for more than 12 days, confirming its good structural stability.

[0050] Another part was used to test the stability of the hydrogel under different physiological conditions. The hydrogel was placed in a 1.5 mL centrifuge tube, and then 1 mL of SGF (pH 1.0), PBS (pH 7.5), SIF (pH 8.5), and H2O2 (1 mM) were added. The samples were then incubated at room temperature. The results are as follows: Figure 7 As shown, when PBS (phosphate buffered saline), SGF (artificial gastric juice), and SIF (artificial intestinal juice) were added, the hydrogel showed no significant changes in a short period of time. However, when 1 mM H2O2 was added, the surface of the hydrogel disintegrated instantly. Furthermore, with increasing time, the hydrogel structure was gradually destroyed in SGF, but the disintegration rate was significantly slower than that in H2O2.

[0051] Example 3: This experimental example demonstrates the rheological properties of the hydrogel prepared in Example 1. Strain measurement using a rheometer The scanning test was conducted at 25°C with a fixed strain of 1% and a frequency scanning range of 0.01 Hz to 10 Hz.

[0052] The results are as follows Figure 8 The results showed that the gel exhibited a high storage modulus (G'). The mechanical properties of the hydrogel (1000 Pa) have surpassed those of most fully recombinant protein hydrogels with a single physical or chemical network.

[0053] Example 4: In vitro cytotoxicity analysis of the hydrogel prepared in Example 1 was performed in this experimental example. To verify the effect of the constructed hydrogel on cell viability, this experiment used CCK8 solution to detect the effect of hydrogels at different times and concentrations (0, 5, 10, 20, 50, 100 μM) on cell viability. MC38 cells were seeded in 96-well plates, with approximately 1 × 10⁶ cells per well. 5 Cells / mL were cultured at 37℃ and 5% CO2 for 24 h. Hydrogels diluted with different concentrations of PBS (0, 5, 10, 20, 50, 100 μM) were added to different replicates and incubated for 12 h. Then, 100 μL of prepared CCK8 solution was added to each well. After 1–4 h, the absorbance at 450 nm was measured using a microplate reader to determine cell viability. The calculation method was: Cell viability (%) = [(Absorbance of experimental wells - Absorbance of blank wells) / (Absorbance of control group - Absorbance of blank wells)] × 100%. Results are as follows: Figure 9 As shown, at a concentration of 100 μM, cell viability remained above 90%, indicating that the constructed hydrogel had no significant cytotoxicity.

[0054] Example 5: This experimental example demonstrates the in vitro targeting properties of the hydrogel prepared in Example 1. Previous studies have reported that RGD can effectively target overexpressed integrin receptors on the cell surface. Therefore, this experiment investigated the potential targeting ability of hydrogels on hydrogen peroxide-induced colon cancer cells (MC38). Cy5 was used as a molecular probe to label Cy5 / RGD-ULDC and Cy5 / TCT. MC38 cells were incubated at 37°C for 24 h in DMEM (Durbeco modified Eagle medium) containing 10% fetal bovine serum, followed by stimulation with hydrogen peroxide (1000 μM) for 2 h. Then, Cy5-Hydrogel (RGD free) and Cy5-Hydrogel (RGD) (in DMEM containing 2% fetal bovine serum) were used instead of DMEM and incubated at 37°C for 1 h. Fresh DMEM was used instead of Cy5 solution, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min, stained with DAPI (4',6-diamidinyl-2-phenylindole) for 10 min, and observed directly under a fluorescence microscope.

[0055] like Figure 10 As shown, the results indicate that the fluorescence signal of Cy5-Hydrogel (RGD) overlaps more with that of MC38 cells than that without RGD, and is distributed on the cell surface, thus demonstrating that Hydrogel-RGD has cell targeting capabilities.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0057] The sequence list is shown below: SEQ ID NO:1: ARGDGTMLPVFCVVEHYENAIEYDCKEEHAEFVLVRKDMLFNQLIEMALLSLGYSHSSAAQAKGLIQVGKWNPVPLSYVTDAPDATVADMLQDVYHVVTLKIQLHSLDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGV GVPGVGVPGVGVPGEGVPGVGVPGVGELGSGLGSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIREREREHHHHHH* SEQ ID NO:2: GCCCGCGGCGATGGCACCATGCTGCCGGTGTTTTGCGTGGTGGAACATTATGAAAACGCGATTGAATATGATTGCAAAGAAGAACATGCGGAATTTGTGCTGGTGCGCAAAGATATGCTGTTTAACCAGCTGATTGAAATGGCGCTGCTGAGCCTGGGCTATAGCCATAGCAGCGCGGCGCAGGCGAAAGGCCTGATTCAGGTGGGCAAATGGAACCCGGTGCCGCTGAGCTATGTGACCGATGCGCCGGATGCGACCGTGGCGGATATGCTGCAGGATGTGTATCATGTGGTGACCCTGAAAATTCAGCTGCATAGCCTGGATGGCCATGGCGTGGGCGTGCCGGGCGTGGGCGTGCCGGGCGTGGGCGTGCCGGGCGAAGGCGTGCCGGGCGTGGGCGTGCCGGGCGTGGGCGTGCCGGGCGTGGGCGTGCCGGGCGTGGGCGTGCCGGGCGAAGGCGTGCCGGGCGTGGGCGTGCCGGGCGTGGGCGTGCCGGGCGTGGGCGTGCCGGGCGTGGGCGTGCCGGGCGAAGGCGTGCCGGGCGTGGGCGTGCCGGGCGTGGGCGAACTGGGCAGCGGCCTGGGCAGCGGCGCGATGGTGGATACCCTGAGCGGCCTGAGCAGCGAACAGGGCCAGAGCGGCGATATGACCATTGAAGAAGATAGCGCGACCCATATTAAATTTAGCAAACGCGATGAAGATGGCAAAGAACTGGCGGGCGCGACCATGGAACTGCGCGATAGCAGCGGCAAAACCATTAGCACCTGGATTAGCGATGGCCAGGTGAAAGATTTTTATCTGTATCCGGGCAAATATACCTTTGTGGAAACCGCGGCGCCGGATGGCTATGAAGTGGCGACCGCGATTACCTTTACCGTGAACGAACAGGGCCAGGTGACCGTGAACGGCAAAGCGACCAAAGGCGATGCGCATATTCGCGAACGCGAACGCGAACATCATCATCATCATCATTGA SEQ ID NO:3: MAHIVMVDAYKPTKLDGHGVGVPGVPGVPGVPGEGVPGVGVPGVPGVGVPGVPGVPGEGVPGVGVPGVPGVGVPGVGVPGEGVPGVGVPGVGELGSGLGSAPQMLRELQETNAALQDVRELLR QQVKEITFLKNTVMESDASKNLNTSVPGVGVPGVPGEGVPGVGPGVPGVPGVPGVGVPGEVPGVGVPGVPGVGVPGVPGVPGEGVPGVPGVGVPGGLLDAHIVMVDAYKPTKLEHHHHHH* SEQ ID NO:4: ATGGCTCACATCGTTATGGTGGACGCCTACAAACCAACCAAACTGGACGGTCACGGTGTTGGTGTTCCAGGTGTTGGTGTACCAGGTGTTGGTGTTCCAGGCGAAGGTGTACCGGGCGTTGGTGTACCGGGTGTTGGTGTACCGGGTGTGGGTGTTCCGGGTGTTGGTGTTCCGGGTGAAGGTGTACCGGGTGTTGGTGTGCCAGGCGTAGGTGTGCCGGGTGTTGGTGTTCCGGGTGTAGGTGTTCCGGGTGAAGGTGTTCCGGGTGTGGGCGTTCCGGGTGTTGGCGAACTGGGCTCTGGTCTGGGCTCCGCACCACAGATGCTGCGTGAACTGCAAGAAACTAACGCGGCGCTGCAAGACGTTCGCGAATTGCTGCGTCAGCAGGTTAAAGAAATCACCTTTCTGAAAAACACTGTGATGGAATCCGATGCATCTAAACTGAACACCTCTGTACCAGGCGTAGGTGTTCCGGGTGTAGGCGTGCCGGGTGAAGGTGTGCCGGGTGTTGGTGTTCCGGGTGTTGGTGTTCCAGGTGTTGGCGTGCCGGGTGTTGGCGTGCCGGGTGAAGGTGTTCCAGGTGTTGGTGTACCGGGTGTTGGTGTACCGGGTGTTGGTGTTCCGGGTGTTGGTGTTCCGGGTGAAGGCGTTCCGGGTGTTGGCGTGCCGGGTGTTGGTGTTCCGGGTGGCCTGCTGGATGCGCACATCGTTATGGTGGACGCGTACAAACCGACCAAACTGGAACACCACCATCACCACCATTGA SEQ ID NO:5: SEQ ID NO:6: SEQ ID NO:7:

Claims

1. A hydrogel based on recombinant proteins, characterized in that, It is formed by the following two fusion proteins through a covalent and non-covalent double cross-linked network: The ULD-SpyCatcher fusion protein, wherein the ULD protein is a cysteine-rich tetrameric protein that is linked to SpyCatcher via an elastin-like protein ELP, and its N-terminus also contains an RGD sequence. The amino acid sequence is shown in SEQ ID NO:

1. The SpyTag-Coil-SpyTag fusion protein, wherein the Coil protein is linked to the SpyTag sequences on both sides by an elastin-like structure, and the amino acid sequence is shown in SEQ ID NO:2; The hydrogel forms a double crosslinked network through SpyTag / SpyCatcher covalent crosslinking, Coil-Coil nonvalent interaction, and ULD tetramer self-assembly.

2. The hydrogel as described in claim 1, characterized in that, The molar mixing ratio of the ULD-SpyCatcher fusion protein and the SpyTag-Coil-SpyTag fusion protein is 1:

2.

3. The hydrogel as described in claim 1, characterized in that, The total protein concentration of the hydrogel is 8 wt%.

4. The hydrogel as described in claim 1, characterized in that, The hydrogel has a pore size of less than 50 micrometers and has a porous structure.

5. The hydrogel according to claim 1, characterized in that, The surface structure of the hydrogel disintegrates within 5 minutes under 1 mM H2O2 conditions, exhibiting reactive oxygen species responsiveness. Preferably, the hydrogel is stable at neutral pH and degrades slowly in acidic or alkaline environments.

6. A method for preparing the hydrogel according to any one of claims 1 to 5, characterized in that, Includes the following steps: Expression vectors were constructed, transformed into host cells and induced to express. The ULD-SpyCatcher and SpyTag-Coil-SpyTag fusion proteins were then purified by Ni-NTA affinity chromatography. The purified ULD-SpyCatcher and SpyTag-Coil-SpyTag fusion proteins were then dialyzed and freeze-dried, and then mixed in phosphate buffer solution for a water bath reaction to obtain the final product.

7. The preparation method according to claim 6, characterized in that, The expression vector is pET32a; the host cell includes Escherichia coli; the conditions for inducing expression are: when the OD600 reaches 0.6~0.8, add IPTG at a final concentration of 0.1 mM, and induce expression at 16℃ for 16-20 h. Preferably, the host cell is Escherichia coli BL21(DE3).

8. The preparation method according to claim 6, characterized in that, The elution buffer for the Ni-NTA affinity chromatography is a 50 mM HEPES buffer containing 50 mM NaCl.

9. The preparation method according to claim 6, characterized in that, The dialysis uses deionized water and the dialysis time is 20-30 hours. The water bath reaction was carried out at a temperature of 37°C for 1 hour.

10. The use of a recombinant protein-based hydrogel according to any one of claims 1 to 5 in drug delivery or tissue engineering.

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