SpyCatcher / SpyTags-polyethylene glycol tissue sealant as well as preparation method and application thereof

Abstract

The invention relates to a SpyCatcher / SpyTags-polyethylene glycol tissue sealant as well as a preparation method and application thereof, and belongs to the technical field of tissue sealants. The preparation method comprises the following steps: sequentially adding SpyCatcher protein, EDTA (Ethylene Diamine Tetraacetic Acid) and 2-imino sulfane hydrochloride into a buffer solution, stirring at 0-4 DEG C overnight, and dialyzing to obtain SpyCatcher-SH; the preparation method comprises the following steps: adding a polyethylene glycol acrylate derivative and SpyCatcher-SH (or SpyTag fusion protein) into a buffer solution, and reacting to obtain a first solution (or a second solution); and mixing and stirring the first solution and the second solution, and adding an enzyme initiation component for catalysis to obtain the product. The SpyCatcher / SpyTag system and the PEG acrylate derivative are catalyzed by the enzyme initiation system to prepare the tissue sealant, so that the problem of poor mechanical property of Spy protein gel is solved, and the potential toxicity of a composite system is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue sealants, and in particular relates to a SpyCatcher / SpyTag-polyethylene glycol tissue sealant and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Skin tissue loss caused by pathological and physiological conditions such as burns and extensive trauma, as well as wound healing difficulties caused by underlying metabolic diseases (such as diabetes and obesity), can hinder normal skin tissue healing. Tissue sealants bind to the surface of biological tissues through electrostatic effects, physical penetration, or covalent bonds, forming a physical barrier that can seal wounds, stop bleeding, or secure transplants.

[0004] SpyCatcher consists of 138 amino acids with a molecular weight of approximately 15 kDa, while SpyTag is a short peptide consisting of only 13 amino acids. SpyCatcher and SpyTag are derived from the immunoglobulin-like collagen adhesion domain (CnaB2) of the fibronectin-binding protein of Streptococcus pyogenes. CnaB2 is cleaved into two parts. Sequence optimization revealed that SpyCatcher and SpyTag can still form a covalent isopeptide bond between Lys31 (SpyCatcher) and Asp117 (SpyTag), while significantly improving the reaction rate. This SpyCatcher / SpyTag protein ligation system has been applied in many fields.

[0005] The existing technology includes the technical solution of expressing SpyCatcher and SpyTag in series with "building block" proteins (such as globular domain GB1, FnIII, C-terminal adenosylcobalamin binding domain CarHC, etc.), and combining them in a "building block" manner to form a gel. It can be used as a cell delivery carrier for disease treatment, but the mechanical properties of the protein colloid are poor, and it is difficult to be used as a tissue sealing material in clinical practice. Among the many gel materials, gels developed based on high molecular weight polyethylene glycol (PEG) (especially PEG acrylate derivative gels, etc.) have excellent mechanical properties, and these derivatives have active groups that can further react with molecules such as proteins to form complex structures, thereby achieving specific functions. However, PEG molecules containing propylene groups usually use photocrosslinking or chemical crosslinking methods to catalyze the formation of covalent bonds between molecules, and crosslinkers are potentially dangerous to tissues and cells and may have side effects. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention aims to provide a SpyCatcher / SpyTag-polyethylene glycol tissue sealant and a preparation method thereof. The tissue sealant is prepared by utilizing the SpyCatcher / SpyTag protein linker system and PEG acrylate derivatives through biomimetic enzyme catalysis. This not only solves the problem of poor mechanical properties of Spy protein gels, but also reduces the potential toxicity of the composite system.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] In a first aspect, a method for preparing a SpyCatcher / SpyTag-polyethylene glycol tissue sealant comprises the steps of:

[0009] S1. SpyCatcher protein, EDTA, and 2-iminothiolane hydrochloride were sequentially added to PBS buffer at a final molar concentration of (0.4-1.4):2:(4-14), and dialyzed to obtain SpyCatcher-SH.

[0010] S2, adding the polyethylene glycol acrylate derivative and the SpyCatcher-SH to a PBS buffer solution at a mass volume concentration ratio of (0.05-0.1): (0.1-10), and reacting to obtain a PEG-SpyCatcher solution;

[0011] S3, adding polyethylene glycol acrylate derivative and SpyTag fusion protein to PBS buffer at a mass volume concentration ratio of (0.05-0.1): (0.02-4) to react to obtain PEG-SpyTag solution;

[0012] S4. After mixing and stirring the PEG-SpyCatcher solution and the PEG-SpyTag solution, an enzyme initiating component is added to the mixed solution to catalyze and obtain a SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0013] In a second aspect, the SpyCatcher / SpyTag-polyethylene glycol tissue sealant prepared by the above preparation method.

[0014] Thirdly, the application of the above-mentioned SpyCatcher / SpyTag-polyethylene glycol tissue sealant in wound repair.

[0015] The beneficial effects of the present invention include:

[0016] This invention proposes, for the first time, the use of a SpyCatcher / SpyTag system and a PEG acrylate derivative in an enzyme-triggered system to prepare a tissue sealant. This approach not only addresses the poor mechanical properties of Spy protein gels but also reduces the potential toxicity of the composite system. The SpyCatcher protein is first thiolated to obtain a reactive -SH group. Subsequently, a thiol-ene click reaction is performed between the SpyCatcher-SH and the N-terminal cysteine-containing SpyTag fusion protein to produce PEG-SpyCatcher and PEG-SpyTag. The PEG-SpyCatcher and PEG-SpyTag are then mixed, where they form covalent bonds through unreacted propylene groups on their molecular chains under the catalysis of the enzyme-triggered system, leading to crosslinking and forming a gel network structure. This structure also incorporates the covalent isopeptide bonds inherent in the SpyCatcher / SpyTag protein linkage system, resulting in a product with high mechanical strength and without the presence of potentially toxic initiators. The resulting tissue sealant exhibits excellent biosafety, tissue adhesion, and mechanical properties, making it suitable for wound repair and other applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 This is a compressive strain-compressive strength test graph of the tissue sealant in Example 4. DETAILED DESCRIPTION

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0020] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0021] The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions. Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available.

[0022] The chemical reagents involved in the specific embodiment include:

[0023] Ethylenediaminetetraacetic acid (EDTA), whose chemical formula is C 10 H 16 N2O8, CAS No. 60-00-4.

[0024] 2-Iminothiolane hydrochloride (Traut's Reagent), CAS No. 4781-83-3.

[0025] Polyethylene glycol diacrylate (PEGDA), CAS No. 26570-48-9.

[0026] Four-arm polyethylene glycol acrylate (4-arm-PEG-Acrl, or 4-arm PEG ACRL) is often used as a modifier for biomaterials.

[0027] Six-arm polyethylene glycol acrylate (6-arm-PEG-Acrl, or 6-arm PEG ACRL) is often used as a modifying agent for biomaterials.

[0028] Eight-arm polyethylene glycol acrylate (8-arm-PEG-Acrl, or 8-arm PEG ACRL) is often used as a modifying agent for biomaterials.

[0029] Ferrous glycinate, molecular formula C4H8FEN2O4, CAS number 20150-34-9.

[0030] Glucose oxidase can catalyze the oxidation of glucose to produce gluconic acid and hydrogen peroxide, while releasing energy.

[0031] CBD-polyD-SpyTag, a SpyTag fusion protein, has an amino acid sequence as shown in SEQ ID NO. 1: CTKKTLRTDDDDDDDDDDDLDDDDDDDDDDDDDDDDDDDDDDDDDDDDDLAHIVMVDAYKPTK.

[0032] CBD-GGGS-SpyTag, SpyTag fusion protein, whose amino acid sequence is shown in SEQ ID NO.2: CTKKTLRTGGSSRSSSSGGGGSGGAHIVMVDAYKPTK.

[0033] Phosphate buffer solution (PBS buffer) was prepared from deionized water, KH2PO4 and Na2HPO4·2H2O.

[0034] The SpyCatcher protein and SpyTag fusion protein used in the method of the present invention can be purchased through commercial channels or obtained by fermentation and purification using genetically engineered bacteria. The specific fermentation and purification method is as follows: BL21 (DE3)-pET28a-SpyCatcher is selected and monocloned into Kan-resistant LB medium (yeast powder 5g / L, NaCl 10g / L, peptone 10g / L) for activation; the next day, the flask is inoculated and cultured, and when the OD 600 When the value was 0.6-0.8, 0.1 mM isopropylthiogalactoside was added for induction for 6 hours, and the cells were collected; the cells were disrupted by high-pressure homogenization, centrifuged at 12000g and 4°C for 10 minutes, the supernatant was discarded, and an equal volume of PBS (0.02M, pH 7.4) was added to resuspend the supernatant; the protein was purified using Ni-NTA; the final collected eluate was dialyzed into PBS (0.02M, pH 7.4) to obtain the SpyCatcher protein. In the above steps, BL21(DE3)-pET28a-SpyCatcher was replaced with BL21(DE3)-pET28a-CBD-linker-SpyTag to obtain the CBD-linker-SpyTag fusion protein.

[0035] A method for preparing a SpyCatcher / SpyTag-polyethylene glycol tissue sealant comprises the following steps:

[0036] S1. SpyCatcher protein, EDTA, and 2-iminothiolane hydrochloride were sequentially added to PBS buffer at a final molar concentration of (0.4-1.4):2:(4-14), and dialyzed to obtain SpyCatcher-SH.

[0037] S2, adding the polyethylene glycol acrylate derivative and the SpyCatcher-SH to a PBS buffer solution at a mass volume concentration ratio of (0.05-0.1): (0.1-10), and reacting to obtain a PEG-SpyCatcher solution (the first solution);

[0038] S3, adding polyethylene glycol acrylate derivative and SpyTag fusion protein to PBS buffer at a mass volume concentration ratio of (0.05-0.1): (0.02-4), and reacting to obtain a PEG-SpyTag solution (the second solution);

[0039] S4. After mixing and stirring the first solution and the second solution, an enzyme initiating component is added to the mixed solution to catalyze the formation of SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0040] Optionally, in S1, the SpyCatcher protein includes a recombinant SpyCatcher protein.

[0041] Optionally, in S1, the final molar concentration of the SpyCatcher protein is 0.4-1.4 mM.

[0042] Optionally, in S1, the reaction process includes: stirring at 0-4°C overnight.

[0043] Optionally, in S1, the molecular weight cut-off for dialysis is 8000-14000 Da.

[0044] Optionally, in S2, the polyethylene glycol acrylate derivative includes any one or more of PEGDA, 4-arm-PEG-Acrl, 6-arm-PEG-Acrl and 8-arm-PEG-Acrl.

[0045] Optionally, in S2, the molecular weight of the polyethylene glycol acrylate derivative is any one or more of 400, 600, 1000, 2000, 3400, 5000 and 10000 Da.

[0046] Optionally, in S2, the SpyCatcher-SH is added to PBS buffer at a mass volume concentration of 0.1 to 10 g / mL.

[0047] Optionally, in S2, after the reaction, ultrafiltration is performed and concentrated to a mass volume concentration of 0.05 to 0.1 g / mL to obtain a PEG-SpyCatcher solution.

[0048] Optionally, in S2, the reaction process includes: reacting at 37±2° C. for 2 to 4 hours.

[0049] Optionally, in S3, the SpyTag fusion protein includes a recombinant SpyTag fusion protein. Preferably, the recombinant SpyTag fusion protein is a fusion protein with cysteine at the N-terminus; more preferably, the recombinant SpyTag fusion protein is a CBD-linker-SpyTag fusion protein with cysteine at the N-terminus, including one or more of CBD-GGGS-SpyTag and CBD-polyD-SpyTag.

[0050] Optionally, in S3, the SpyTag fusion protein is added to PBS buffer at a mass volume concentration of 0.02 to 4 g / mL.

[0051] Optionally, in S3, after the reaction, ultrafiltration is performed and concentrated to a mass volume concentration of 0.05 to 0.1 g / mL to obtain a PEG-SpyTag solution.

[0052] Optionally, in S3, the reaction process includes: reacting at 37±2° C. for 2 to 4 hours.

[0053] Optionally, in S4, the enzyme initiating component includes glucose oxidase, ferrous glycine and glucose, which is used to catalyze the unreacted propylene groups on the molecular chains of the two to form covalent bonds and cross-link to form a gel network structure.

[0054] Optionally, the dosage of glucose oxidase in the mixed solution is 10-100 U, the concentration of ferrous glycine is 0.1-5 mM, and the concentration of glucose is 0.2-10 mM.

[0055] Optionally, the dosage of the glucose oxidase is 50-60 U, the concentration of ferrous glycine is 1-1.5 mM, and the concentration of glucose is 1-2 mM.

[0056] The SpyCatcher / SpyTag-polyethylene glycol tissue sealant prepared by the above preparation method.

[0057] The use of the above-mentioned SpyCatcher / SpyTag-polyethylene glycol tissue sealant in skin wound repair.

[0058] Optionally, in actual applications, the solutions obtained from S2 and S3 are mixed, and then glucose oxidase and ferrous glycine are added to form a premixed solution, and the premixed solution is directly applied to the wound; since there is a low concentration of glucose in the wound, it can automatically trigger the formation of a colloid for wound repair, and there is no need to add glucose to trigger the gelation reaction.

[0059] Example 1

[0060] A SpyCatcher / SpyTag-polyethylene glycol tissue sealant, the preparation method comprising:

[0061] S1. Dissolve the SpyCatcher protein in PBS at a molar concentration of 0.4 mM. Add EDTA to a final concentration of 2 mM and 2-iminothiolane hydrochloride (Traut's Reagent) to a final concentration of 4 mM to obtain a mixed liquid. Stir the reaction system at 4°C overnight. Then, use a dialysis bag in PBS to retain molecules with a molecular weight cutoff of 8,000-14,000 Da to obtain SpyCatcher-SH.

[0062] S2. Dissolve PEGDA with a molecular weight of 400 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyCatcher-SH to a mass volume concentration of 0.1 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyCatcher solution with a mass volume concentration of 0.05 g / mL.

[0063] S3. Dissolve PEGDA with a molecular weight of 400 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyTag fusion protein (CBD-GGGS-SpyTag) to a mass volume concentration of 0.02 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyTag solution with a mass volume concentration of 0.05 g / mL.

[0064] S4. Mix equal volumes of PEG-SpyCatcher solution and PEG-SpyTag solution and stir to obtain a mixed liquid. Add 10 U of glucose oxidase and ferrous glycine to a molar concentration of 0.1 mM to the mixed liquid. After mixing, add glucose to a molar concentration of 0.2 mM to catalyze the formation of SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0065] Its compressive strength is 0.23 MPa and its gelation time is 6 seconds. The gelation time refers to the time from the addition of glucose to the complete conversion of the mixed liquid into a gel state.

[0066] Example 2

[0067] A SpyCatcher / SpyTag-polyethylene glycol tissue sealant, the preparation method comprising:

[0068] S1. Dissolve the SpyCatcher protein in PBS at a molar concentration of 1.4 mM. Add EDTA to a final concentration of 2 mM and 2-iminothiolane hydrochloride (Traut's Reagent) to a final concentration of 14 mM to obtain a mixed liquid. Stir the reaction system at 4°C overnight. Then, use a dialysis bag in PBS to retain substances with a molecular weight cutoff of 8000-14000 Da to obtain SpyCatcher-SH.

[0069] S2. Dissolve 4-arm-PEG-Acrl with a molecular weight of 600 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyCatcher-SH to a mass volume concentration of 0.4 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyCatcher solution with a mass volume concentration of 0.05 g / mL.

[0070] S3. Dissolve 4-arm-PEG-Acrl with a molecular weight of 600 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyTag fusion protein (CBD-GGGS-SpyTag) to a mass volume concentration of 0.1 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyTag solution with a mass volume concentration of 0.05 g / mL.

[0071] S4. Mix equal volumes of PEG-SpyCatcher solution and PEG-SpyTag solution and stir to obtain a mixed liquid. Add 10 U of glucose oxidase and ferrous glycine to a molar concentration of 0.1 mM to the mixed liquid. After mixing, add glucose to a molar concentration of 2 mM to catalyze the formation of SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0072] Its compressive strength is 0.42MPa and gelation time is 4.2s.

[0073] Example 3

[0074] A SpyCatcher / SpyTag-polyethylene glycol tissue sealant, the preparation method comprising:

[0075] S1. Dissolve the SpyCatcher protein in PBS at a molar concentration of 1.4 mM. Add EDTA to a final concentration of 2 mM and 2-iminothiolane hydrochloride (Traut's Reagent) to a final concentration of 14 mM to obtain a mixed liquid. Stir the reaction system at 4°C overnight. Then, use a dialysis bag in PBS to retain substances with a molecular weight cutoff of 8,000-14,000 Da to obtain SpyCatcher-SH.

[0076] S2. Dissolve 6-arm-PEG-Acrl with a molecular weight of 1000 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyCatcher-SH to a mass volume concentration of 0.6 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyCatcher solution with a mass volume concentration of 0.05 g / mL.

[0077] S3. Dissolve 6-arm-PEG-Acrl with a molecular weight of 1000 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyTag fusion protein (CBD-polyD-SpyTag) to a mass volume concentration of 0.15 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyTag solution with a mass volume concentration of 0.05 g / mL.

[0078] S4. Mix equal volumes of PEG-SpyCatcher solution and PEG-SpyTag solution and stir to obtain a mixed liquid. Add 10 U of glucose oxidase and ferrous glycine to a molar concentration of 0.1 mM to the mixed liquid. After mixing, add glucose to a molar concentration of 5 mM to catalyze the formation of SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0079] Its compressive strength is 0.61MPa and the gelation time is 2.6s.

[0080] Example 4

[0081] A SpyCatcher / SpyTag-polyethylene glycol tissue sealant, the preparation method comprising:

[0082] S1. Dissolve the SpyCatcher protein in PBS at a molar concentration of 1.4 mM. Add EDTA to a final concentration of 2 mM and 2-iminothiolane hydrochloride (Traut's Reagent) to a final concentration of 14 mM to obtain a mixed liquid. Stir the reaction system at 4°C overnight. Then, use a dialysis bag in PBS to retain substances with a molecular weight cutoff of 8,000-14,000 Da to obtain SpyCatcher-SH.

[0083] S2. Dissolve 8-arm-PEG-Acrl with a molecular weight of 1000 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyCatcher-SH to a mass volume concentration of 10 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyCatcher solution with a mass volume concentration of 0.1 g / mL.

[0084] S3. Dissolve 8-arm-PEG-Acrl with a molecular weight of 1000 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyTag fusion protein (CBD-polyD-SpyTag) to a mass volume concentration of 4 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyTag solution with a mass volume concentration of 0.1 g / mL.

[0085] S4. Mix equal volumes of PEG-SpyCatcher solution and PEG-SpyTag solution and stir to obtain a mixed liquid. Add 50 U of glucose oxidase and ferrous glycine to the mixed liquid to a molar concentration of 5 mM. After mixing, add glucose to a molar concentration of 10 mM to catalyze the formation of SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0086] Its compressive strength is 1.69MPa and the gelation time is less than 1s.

[0087] Example 5

[0088] A SpyCatcher / SpyTag-polyethylene glycol tissue sealant, the preparation method comprising:

[0089] S1. Dissolve the SpyCatcher protein in PBS at a molar concentration of 1.4 mM. Add EDTA to a final concentration of 2 mM and 2-iminothiolane hydrochloride (Traut's Reagent) to a final concentration of 14 mM to obtain a mixed liquid. Stir the reaction system at 4°C overnight. Then, use a dialysis bag in PBS to retain substances with a molecular weight cutoff of 8,000-14,000 Da to obtain SpyCatcher-SH.

[0090] S2. Dissolve 8-arm-PEG-Acrl with a molecular weight of 2000 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyCatcher-SH to a mass volume concentration of 5 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyCatcher solution with a mass volume concentration of 0.05 g / mL.

[0091] S3. Dissolve 8-arm-PEG-Acrl with a molecular weight of 2000 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyTag fusion protein (CBD-polyD-SpyTag) to a mass volume concentration of 2 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyTag solution with a mass volume concentration of 0.05 g / mL.

[0092] S4. Mix equal volumes of PEG-SpyCatcher solution and PEG-SpyTag solution and stir to obtain a mixed liquid. Add 50 U of glucose oxidase and ferrous glycine to the mixed liquid to a molar concentration of 3 mM. After mixing, add glucose to a molar concentration of 1 mM to catalyze the formation of SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0093] Its compressive strength is 0.74MPa and its gelling time is 2.7s.

[0094] Example 6

[0095] A SpyCatcher / SpyTag-polyethylene glycol tissue sealant, the preparation method comprising:

[0096] S1. Dissolve the SpyCatcher protein in PBS at a molar concentration of 1.4 mM. Add EDTA to a final concentration of 2 mM and 2-iminothiolane hydrochloride (Traut's Reagent) to a final concentration of 14 mM to obtain a mixed liquid. Stir the reaction system at 4°C overnight. Then, use a dialysis bag in PBS to retain substances with a molecular weight cutoff of 8,000-14,000 Da to obtain SpyCatcher-SH.

[0097] S2. Dissolve 8-arm-PEG-Acrl with a molecular weight of 3400 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyCatcher-SH to a mass volume concentration of 1 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyCatcher solution with a mass volume concentration of 0.1 g / mL.

[0098] S3. Dissolve 8-arm-PEG-Acrl with a molecular weight of 3400 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyTag fusion protein (CBD-polyD-SpyTag) to a mass volume concentration of 0.5 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyTag solution with a mass volume concentration of 0.1 g / mL.

[0099] S4. Mix equal volumes of PEG-SpyCatcher solution and PEG-SpyTag solution and stir to obtain a mixed liquid. Add glucose oxidase to 100 U and ferrous glycine to a molar concentration of 5 mM to the mixed liquid. After mixing, add glucose to a molar concentration of 5 mM to catalyze the formation of SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0100] Its compressive strength is 1.13MPa and its gelling time is 1.9s.

[0101] Example 7

[0102] A SpyCatcher / SpyTag-polyethylene glycol tissue sealant, the preparation method comprising:

[0103] S1. Dissolve the SpyCatcher protein in PBS at a molar concentration of 1.4 mM. Add EDTA to a final concentration of 2 mM and 2-iminothiolane hydrochloride (Traut's Reagent) to a final concentration of 14 mM to obtain a mixed liquid. Stir the reaction system at 4°C overnight. Then, use a dialysis bag in PBS to retain substances with a molecular weight cutoff of 8,000-14,000 Da to obtain SpyCatcher-SH.

[0104] S2. Dissolve 6-arm-PEG-Acrl with a molecular weight of 5000 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyCatcher-SH to a mass volume concentration of 3 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyCatcher solution with a mass volume concentration of 0.1 g / mL.

[0105] S3. Dissolve 6-arm-PEG-Acrl with a molecular weight of 5000 in PBS solution to a mass volume concentration of 0.05 g / mL, then add SpyTag fusion protein (CBD-polyD-SpyTag) to a mass volume concentration of 1 g / mL, react at 37°C for 2 h, and concentrate by ultrafiltration to obtain a PEG-SpyTag solution with a mass volume concentration of 0.1 g / mL.

[0106] S4. Mix equal volumes of PEG-SpyCatcher solution and PEG-SpyTag solution and stir to obtain a mixed liquid. Add 10 U of glucose oxidase and ferrous glycine to a molar concentration of 0.1 mM to the mixed liquid. After mixing, add glucose to a molar concentration of 1 mM to catalyze the formation of SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

[0107] Its compressive strength is 1.48MPa and its gelling time is 4.4s.

[0108] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a SpyCatcher / SpyTag-polyethylene glycol tissue sealant, characterized in that: Including steps: S1. SpyCatcher protein, EDTA, and 2-iminothiolane hydrochloride were sequentially added to PBS buffer at a final molar concentration of (0.4-1.4):2:(4-14), and dialyzed to obtain SpyCatcher-SH. S2, adding the polyethylene glycol acrylate derivative and the SpyCatcher-SH to a PBS buffer solution at a mass volume concentration ratio of (0.05-0.1): (0.1-10), and reacting to obtain a PEG-SpyCatcher solution; S3, adding polyethylene glycol acrylate derivative and SpyTag fusion protein to PBS buffer at a mass volume concentration ratio of (0.05-0.1): (0.02-4) to react to obtain PEG-SpyTag solution; S4. After mixing and stirring the PEG-SpyCatcher solution and the PEG-SpyTag solution, an enzyme initiating component is added to the mixed solution to catalyze and obtain a SpyCatcher / SpyTag-polyethylene glycol tissue sealant.

2. The method for preparing the SpyCatcher / SpyTag-polyethylene glycol tissue sealant according to claim 1, wherein: In S1, the SpyCatcher protein includes a recombinant SpyCatcher protein; or, in S1, the final molar concentration of the SpyCatcher protein is 0.4 to 1.4 mM; Alternatively, in S1, the reaction process comprises: stirring at 0-4°C overnight; Alternatively, in S1, the molecular weight cut-off of the dialysis method is 8000-14000 Da.

3. The method for preparing the SpyCatcher / SpyTag-polyethylene glycol tissue sealant according to claim 1, wherein: In S2, the polyethylene glycol acrylate derivative includes any one or more of PEGDA, 4-arm-PEG-Acrl, 6-arm-PEG-Acrl and 8-arm-PEG-Acrl; Or in S2, the molecular weight of the polyethylene glycol acrylate derivative is any one or more of 400, 600, 1000, 2000, 3400, 5000 and 10000 Da.

4. The method for preparing the SpyCatcher / SpyTag-polyethylene glycol tissue sealant according to claim 1, wherein: In S2, the SpyCatcher-SH is added to PBS buffer at a mass volume concentration of 0.1 to 10 g / mL; Alternatively, in S2, after the reaction, ultrafiltration is performed and concentrated to a mass volume concentration of 0.05 to 0.1 g / mL to obtain a PEG-SpyCatcher solution; Alternatively, in S2, the reaction process includes: reacting at 37±2° C. for 2 to 4 hours.

5. The method for preparing the SpyCatcher / SpyTag-polyethylene glycol tissue sealant according to claim 1, wherein: In S3, the SpyTag fusion protein includes a recombinant SpyTag fusion protein; Or, the recombinant SpyTag fusion protein is a fusion protein with cysteine at the N-terminus; Alternatively, the recombinant SpyTag fusion protein is a CBD-linker-SpyTag fusion protein with cysteine at the N-terminus, one or more of CBD-GGGS-SpyTag and CBD-polyD-SpyTag, the amino acid sequence of CBD-GGGS-SpyTag is shown in SEQ ID NO.2; the amino acid sequence of CBD-polyD-SpyTag is shown in SEQ ID NO.

1.

6. The method for preparing the SpyCatcher / SpyTag-polyethylene glycol tissue sealant according to claim 1, wherein: In S3, the SpyTag fusion protein is added to PBS buffer at a mass volume concentration of 0.02 to 4 g / mL; Alternatively, in S3, after the reaction, ultrafiltration is performed and concentrated to a mass volume concentration of 0.05 to 0.1 g / mL to obtain a PEG-SpyTag solution; Alternatively, in S3, the reaction process includes: reacting at 37±2° C. for 2 to 4 hours.

7. The method for preparing the SpyCatcher / SpyTag-polyethylene glycol tissue sealant according to claim 1, wherein: In S4, the enzyme initiating component includes glucose oxidase, ferrous glycine and glucose.

8. The method for preparing the SpyCatcher / SpyTag-polyethylene glycol tissue sealant according to claim 7, wherein: The mixed solution comprises 10 to 100 U of glucose oxidase, 0.1 to 5 mM of ferrous glycine, and 0.2 to 10 mM of glucose. Alternatively, the dosage of the glucose oxidase is 50-60 U, the concentration of ferrous glycine is 1-1.5 mM, and the concentration of glucose is 1-2 mM.

9. A SpyCatcher / SpyTag-polyethylene glycol tissue sealant prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the SpyCatcher / SpyTag-polyethylene glycol tissue sealant according to claim 9 in skin wound repair.

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