A method for expressing vascular endothelial growth factor by using a gel type wheat germ cell-free protein system and use thereof

Abstract

The present application belongs to the field of biotechnology, and relates to a method for expressing vascular endothelial growth factor by a gel type wheat germ cell-free protein system and application. The present application selects carboxymethyl chitosan and hyaluronic acid as raw materials, and after formula optimization of mechanical and biocompatibility parameters, the components of the cell-free protein synthesis system are integrated into the double network architecture, forming a system capable of realizing sustained vascular endothelial growth factor expression through encapsulated mRNA synthesis, which effectively relieves the burst toxicity, and realizes controlled treatment delivery. In addition, the open nature of the cell-free protein synthesis system allows programmable biosynthesis according to nucleic acid templates, thereby expressing various functional proteins, which greatly expands the potential of its transformation application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the preparation of vascular endothelial growth factor. Background Technology

[0002] The skin, the largest organ in the human body, plays a crucial role in barrier function, thermoregulation, sensory perception, immune defense, and metabolic activities. Skin injury disrupts this barrier function, allowing pathogens to invade, leading to wound infection and tissue damage. These infections prolong the inflammatory phase, reduce oxygenation levels in deep tissues, inhibit fibroblast proliferation, interfere with angiogenesis, and hinder epithelial regeneration. Such impairments prolong healing time, increase the medical burden, and worsen the patient's condition. Severe cases may progress to life-threatening systemic complications.

[0003] Current single-mode antimicrobial strategies, including antibiotics, nanoparticles, and cationic polymers, have limitations. Antibiotic resistance is widespread, and broad-spectrum antibiotics disrupt symbiotic microbiota and pathogens. More importantly, most antimicrobial methods neglect key healing processes such as inflammation resolution and tissue regeneration. Therefore, multifunctional dressings with both antimicrobial and regenerative properties are crucial. An ideal dressing should maintain a moist microenvironment, prevent bacterial invasion, and possess biocompatibility and bioactive healing-promoting effects.

[0004] Hydrogels, as highly absorbent three-dimensional polymer networks, exhibit great potential by maintaining structural integrity even after absorbing water. Their moisturizing properties promote cell migration / proliferation and accelerate wound healing. The viscoelasticity of hydrogels allows them to adapt to wound topography, thereby reducing shear stress. Carboxymethyl chitosan, a water-soluble chitosan derivative, possesses biocompatibility, biodegradability, and hemostatic properties. It forms a protective barrier against pathogens, absorbs exudate to maintain moisture, and supports cellular activity. Hyaluronic acid, an endogenous glycosaminoglycan, enhances moisturizing, promotes cell migration / proliferation, regulates inflammation, and provides a structural scaffold for tissue regeneration. These properties make carboxymethyl chitosan and hyaluronic acid ideal choices for hydrogel dressings.

[0005] Angiogenesis, the process of delivering nutrients and oxygen through the formation of new blood vessels, is a potential therapeutic strategy for wound healing in infected patients. Vascular endothelial growth factor (VEGF) is a powerful signaling molecule that drives angiogenesis by promoting the proliferation, migration, and differentiation of endothelial cells into new blood vessels. Recombinant human VEGF protein therapy, particularly the VEGF-A165 subtype, has demonstrated significant clinical efficacy in vascular repair. As an alternative to recombinant proteins, gene-based therapies (utilizing DNA or mRNA) have attracted widespread attention. Cell-free protein synthesis systems enable in vitro protein expression using exogenous mRNA or DNA templates, enzymes, amino acid substrates, and energy sources. Unlike traditional in vivo recombinant expression, cell-free protein synthesis systems allow direct control of reaction components, avoid product toxicity to host cells, and offer ease of operation—qualities that facilitate functional protein research. Publication number CN 113336839 A discloses a method for expressing human fibroblast growth factor 21 using a wheat germ cell-free protein synthesis system. This method primarily involves constructing an expression vector and then expressing it in the wheat germ cell-free protein synthesis system, mainly for the preparation of recombinant proteins. Traditional solution-phase cell-free protein synthesis systems (tubular or reactor-based) inherently lack spatial control, resulting in low protein synthesis efficiency, poor protein stability, and difficulty in conducting long-term functional studies. (Disadvantages) In contrast, gel-based wheat germ cell-free systems, through gel immobilization technology, effectively improve the stability of the reaction system and the efficiency of protein synthesis. Compared to traditional liquid systems, their advantages lie in the ability to maintain protein and reaction activity for extended periods, and hydrogels possess biocompatibility, tissue repair, and drug delivery properties. Recent studies have demonstrated the feasibility of encapsulating genetic information (circular / linear DNA), transcription-translation enzyme systems, and energy regeneration systems within a hydrogel matrix. This method can prepare porous biomaterial-based artificial cells, enabling long-term, continuous protein secretion. However, the potential for synergistic application of functional proteins generated by cell-free protein synthesis systems with hydrogel systems to promote cell proliferation and wound repair remains unexplored. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a method and application for expressing vascular endothelial growth factor (VEGF) using a gel-type wheat germ cell-free protein system. This invention uses carboxymethyl chitosan and hyaluronic acid as raw materials. After optimizing the formulation of mechanical and biocompatibility parameters, the components of the cell-free protein synthesis system are integrated into this dual-network architecture, forming a system capable of sustained VEGF expression through encapsulated mRNA synthesis. This effectively mitigates burst-release toxicity and achieves controlled therapeutic delivery. Furthermore, the open nature of the cell-free protein synthesis system allows for programmable biosynthesis based on nucleic acid templates, thereby expressing various functional proteins, which greatly expands its translational application potential.

[0007] The technical solution of this invention is implemented as follows:

[0008] On one hand, the present invention provides a method for expressing vascular endothelial growth factor in a gel-type wheat germ cell-free protein system, the steps of which are: thiolated carboxymethyl chitosan to generate thiolated carboxymethyl chitosan; oxidized hyaluronic acid to generate oxidized hyaluronic acid; modified oxidized hyaluronic acid with maleimide to generate maleimide-based oxidized hyaluronic acid; and encapsulated the cell-free protein synthesis system in a two-component hydrogel.

[0009] The degree of substitution of carboxymethyl chitosan in the two-component hydrogel is >90%, and the molecular weight of hyaluronic acid is 800,000.

[0010] In the above two-component hydrogel, maleimide-oxidized hyaluronic acid and thiolated carboxymethyl chitosan were both prepared with deionized water at a concentration of 3%~5% (w / v).

[0011] The above-mentioned two-component hydrogel is mixed at room temperature with maleimide-oxidized hyaluronic acid and thiolated carboxymethyl chitosan in a ratio of 1:2 to 1:4.5. The carboxymethyl chitosan has a carboxyl substitution degree >80%, preferably over 90%; the hyaluronic acid has a molecular weight of 400,000 to 800,000, preferably 800,000.

[0012] The nucleic acid sequences used in the construction of the above cell-free protein synthesis system are SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8.

[0013] The above nucleic acid sequences were extended and ligated to form SEQ ID No.7 + SEQ ID No.1 / 2 / 3 / 4 / 5 / 6 + SEQ ID No.8.

[0014] The above ligation products were transcribed into mRNA in vitro, and a DNA tag encoding 6 histidine residues was added to the tail of each mRNA.

[0015] The above-mentioned in vitro transcription buffer was prepared with 400 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 80 mM magnesium acetate, 10 mM spermidine, and 50 mM dithiothreitol.

[0016] In the construction of the cell-free protein synthesis system, whole wheat germ is crushed according to the shaver shaft spacing (700~800 μm).

[0017] In the construction of the cell-free protein synthesis system, whole wheat germ was screened according to the condition of cyclohexane:carbon tetrachloride (V:V) = 2:5.

[0018] In the construction of the cell-free protein synthesis system, the wheat germ extract was prepared with 80 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 200 mM potassium acetate, 10 mM magnesium acetate, and 4 mM calcium chloride.

[0019] In the construction of the cell-free protein synthesis system, the translation supplement solution was prepared according to 120 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 400 mM potassium acetate, 10.8 mM magnesium acetate, 1.6 mM spermidine, 10 mM dithiothreitol, 1.2 mM amino acid standard solution, 4.8 mM adenosine triphosphate, 1 mM guanosine triphosphate, and 64 mM creatine phosphate.

[0020] In the multifunctional two-component hydrogel that encapsulates the cell-free protein synthesis system described above, the solvent for 3%~5% (w / v) maleimide-based oxidized hyaluronic acid and thiolated carboxymethyl chitosan prepared with deionized water is replaced with 1× translation supplement.

[0021] The mRNA of vascular endothelial growth factor was encapsulated in a two-component hydrogel prepared with 1× translation supplementation solution. In step (2) above, the volume ratio of template mRNA, wheat germ extract and phosphokinase was 1:1:0.05; the concentration of phosphokinase was 20 mg / mL; the concentration of template mRNA was ≥1 μg / μL; and the A260 value of wheat germ extract was 95~105.

[0022] Secondly, vascular endothelial growth factor prepared using the above-described method is provided.

[0023] Thirdly, the application of the aforementioned vascular endothelial growth factor in the preparation of functional proteins or products for healing infected wounds is provided.

[0024] The present invention has the following beneficial effects:

[0025] 1. This invention discloses a method and application for expressing vascular endothelial growth factor (VEGF) using a gel-type cell-free wheat germ protein system. The system comprises a cell-free wheat germ system and a dual-network hydrogel. The cell-free wheat germ system is constructed from common hard wheat. The dual-network hydrogel is prepared by dissolving thiol-modified carboxymethyl chitosan and maleimide-modified oxidized hyaluronic acid separately in a translation supplement solution. The gel-type cell-free wheat germ protein system, obtained by uniformly distributing the cell-free system within the dual-network hydrogel, achieves the continuous synthesis and release of VEGF in vitro. In this composite system, the dual-network hydrogel exhibits high antioxidant bioactivity and antibacterial properties. The VEGF expressed in the cell-free system also promotes cell migration and proliferation, accelerating the healing process of infected wounds. Notably, the gel-type cell-free wheat germ system possesses high flexibility and editability, allowing for flexible adaptation to various therapeutic protein genes based on different functional protein genes. This expands its application to multiple fields such as tissue regeneration, anti-inflammatory regulation, and tumor treatment, providing broad prospects for precision medicine.

[0026] 2. This invention is the first to encapsulate a cell-free protein synthesis system within a multifunctional two-component OM / CS hydrogel, enabling in-situ synthesis and release of bioactive vascular endothelial growth factor. This invention improves the biocompatibility of the OM / CS hydrogel, promoting cell proliferation, migration, and wound healing in infected areas. The preparation method of this invention is simple, and the materials are widely available, making it suitable for widespread application. This invention establishes new design principles for the biomedical applications of advanced wound dressings and cell-free protein synthesis systems. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Images of wheat germ screening and acquisition in this invention are shown; where A represents wheat; B represents the wheat germ separation effect; C represents wheat germ; and D represents the extracted mixture.

[0029] Figure 2 The images show the separation of the expression template used in the cell-free wheat germ system constructed in this invention by 1% agarose gel electrophoresis; where A represents DNA and B represents mRNA.

[0030] Figure 3These are laser confocal microscopy images and Western blot images of the cell-free wheat germ system constructed in this invention expressing fluorescent proteins in vitro; where A is the laser confocal result of green fluorescent protein (sfGFP protein) and cherry red fluorescent protein (mCherry protein), scale bar: 10 μm; B is the Western blot result of vitreous hemoglobin, legume hemoglobin, green fluorescent protein, porcine hemoglobin, and human brain nerve growth factor; C is the relative expression level of the five groups of proteins.

[0031] Figure 4 The diagram shows the results of the gel-type cell-free protein synthesis system in this invention; where A represents the protein expression results and B represents the release rate results.

[0032] Figure 5 The results are for testing hyaluronic acid, oxidized hyaluronic acid, maleimide-based oxidized hyaluronic acid, carboxymethyl chitosan, thiolated carboxymethyl chitosan, and gel in this invention; where A is infrared spectroscopy and B is nuclear magnetic resonance hydrogen spectroscopy.

[0033] Figure 6 The results are the test results of hyaluronic acid, oxidized hyaluronic acid, maleimide-based oxidized hyaluronic acid, carboxymethyl chitosan, thiolated carboxymethyl chitosan and gel in this invention; where A is thermogravimetric analysis and B is differential thermal scanning.

[0034] Figure 7 The X-ray diffraction test results of hyaluronic acid, oxidized hyaluronic acid, maleimide-based oxidized hyaluronic acid, carboxymethyl chitosan, thiolated carboxymethyl chitosan and gel in this invention are shown.

[0035] Figure 8 The images show the scanning electron microscopy (SEM) results of gels with different ratios in this invention; where A is OM solution; B is CS solution; C is OM / CS gel; D is SEM images of gels with different ratios, af represents gels of 1:4.5-1:2, and the scale bar is 50 μm.

[0036] Figure 9 These are the rheological test results of hydrogels with different proportions in this invention; where A represents the rheological properties of hydrogels with different proportions under shear strain of 1%-100%; and B represents the rheological properties of hydrogels with different proportions under angular frequencies of 1-40 rad / s.

[0037] Figure 10 The results show the dye release and adhesion of the hydrogel at different pH values ​​in this invention; where A represents the in-situ gelation of the hydrogel on the pigskin surface under different directional forces such as torsion, bending, compression, and stretching; and B represents the effect of different pH solutions on the adhesion and dye release of the hydrogel.

[0038] Figure 11Different proportions of hydrogels in this invention S. aureus , P. aeruginosa and E. coli The growth inhibition result; where A is P. aeruginosa The growth curve; B is S. aureus The growth curve; C is E. coli The growth curve; D is the colony count and live / dead cell image, scale bar: 20 μm.

[0039] Figure 12 The results show the biocompatibility test results of the gel and cell-free protein expression system in this invention; where A represents cell morphology, scale bar: 200 μm; B represents live and dead cell staining, scale bar: 200 μm; C represents Huvec cell viability results; and D represents Hsf cell viability results.

[0040] Figure 13 These are representative images of cell proliferation in the gel and cell-free protein expression systems of this invention; where A represents Huvec cells and B represents Hsf cells. Scale bar: 200 μm.

[0041] Figure 14 These are representative images of scratch area growth in the gel and cell-free protein expression systems of this invention; where A represents Huvec cells and B represents Hsf cells. Scale bar: 200 μm.

[0042] Figure 15 The results of the gel and cell-free protein expression system in this invention promoting wound healing in mice are shown below; A shows representative images of the wounds on days 3, 6, 9 and 12; B shows the statistical analysis of the wound area; C shows the simulated in vivo wound closure curves for different treatments; D shows the hematoxylin and eosin staining of wound skin tissue collected from different groups on day 12, scale bar: 500 μm. Detailed Implementation

[0043] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0044] In this application, the full English name of carboxymethyl chitosan is CMCS. The full English name of hyaluronic acid is HA. The full English name of thiolated carboxymethyl chitosan is CS. The full English name of oxidized hyaluronic acid is OHA. The full English name of maleimide-oxidized hyaluronic acid is OM. The multifunctional hydrogel is obtained by mixing OM and CS in different proportions, and is therefore referred to as OM / CS hydrogel (Gel). The full English name of cell-free protein expression system is CFPS (CF). The full English name of vascular endothelial growth factor is VEGF.

[0045] The primer information used in this application is as follows:

[0046]

[0047] Example 1: Preparation of a multifunctional two-component hydrogel

[0048] In this embodiment, the multifunctional two-component hydrogel was prepared by using carboxymethyl chitosan and hyaluronic acid as raw materials and modifying them. The steps are as follows:

[0049] Step 1: Carboxymethyl chitosan (approximately 20 mM) was completely dissolved in deionized water. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (30 mM) and L-cysteine ​​hydrochloride (30 mM) were added, and the mixture was allowed to react at room temperature for 24 h. After the reaction was complete, the product was dialyzed in 0.01 M borate buffer for 3 days, with multiple water changes during the process. After dialyzing, the dialysate was treated with dithiothreitol and then freeze-dried. The freeze-dried thiolized chitosan (CS) was added to anhydrous ethanol, stirred at 4 °C for 1 h, and then filtered under vacuum.

[0050] Step 2, Synthesis of Oxidized Hyaluronic Acid: Hyaluronic acid (molecular weight 400,000~800,000) was dissolved in 200 mL of distilled water at 4 °C overnight, and then an equimolar amount of sodium periodate was added with stirring. The reaction was carried out in the dark at room temperature for 24 h. After the reaction was completed, an equimolar amount of ethylene glycol was added to the reaction solution to quench unreacted sodium periodate. The obtained product was dialyzed with distilled water for 3 days to remove byproducts. Finally, the oxidized hyaluronic acid solution was lyophilized for 48 h to obtain a powder.

[0051] Step 3: Dissolve oxidized hyaluronic acid in 200 mL of distilled water at room temperature. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (10 mM) and N-hydroxysuccinimide (10 mM) (Shanghai Macklin) to the reaction mixture, and stir at room temperature for 2 h to activate the carboxyl groups on the oxidized hyaluronic acid. After the reaction is complete, add N-(2-aminoethyl)maleimide hydrochloride (10 mM) (Shanghai Macklin) to the system and react at room temperature for 24 h at pH 5.5. Maleimide-based oxidized hyaluronic acid can be prepared by reacting oxidized hyaluronic acid monomer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and N-(2-aminoethyl)maleimide in a molar ratio ranging from 1:2 to 3:2 to 3:2 to 3:2 to 3. A ratio of 1:2.5:2.5:2.5 was used as an example. After the reaction, the resulting product was dialyzed against 0.01 M HCl solution for 3 days to remove byproducts (this process can be achieved within a concentration range of 0.01 M to 0.05 M). Finally, the maleimide-based oxidized hyaluronic acid solution was lyophilized for 48 h until it reached a sponge-like state.

[0052] Step 4: Dissolve thiolated chitosan in water to prepare a 5 wt% thiolated chitosan solution, denoted as CS solution. Dissolve maleimide-based oxidized hyaluronic acid powder in water to prepare a 5 wt% maleimide-based oxidized hyaluronic acid solution, denoted as maleimide-based oxidized hyaluronic acid solution.

[0053] Step 5: Add 5 wt% of thiolated carboxymethyl chitosan and 5 wt% of maleimide-based oxidized hyaluronic acid sequentially to centrifuge tubes according to volume ratios of 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, and 1:2. Vortex for 3 seconds and let stand for a few seconds to obtain a multifunctional bicomponent hydrogel. The mass percentage of thiolated carboxymethyl chitosan and maleimide-based oxidized hyaluronic acid in this step can be 3-5%.

[0054] Example 2: Preparation of a cell-free protein expression system from wheat germ

[0055] This example uses Xinmai 26 wheat variety as raw material to construct a cell-free protein expression system from wheat germ. The specific steps are as follows:

[0056] Step 1, Obtaining wheat germ: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] Figure 1(A) Pour the wheat into a grinder at a flow rate of 100 g / min, and adjust the grinder's shaft distance (700~800 μm) to ensure the ground wheat particles contain a high proportion of wheat germ. Sequentially sieve the sample using mesh sizes (25, 35, 45, 55, 65, 75, 80), manually selecting intact wheat germ particles from each mesh. Soak the intact wheat germ in a cyclohexane / carbon tetrachloride mixed solution to collect the floating particles. Figure 1 (B), then the suspended particles were placed on filter paper in a fume hood to remove the solvent overnight, and those yellow particles containing a small amount of white matter were selected with a toothpick. Figure 1 (C)

[0057] Step 2, Preparation of wheat germ extract: Wash the wheat germ with pre-cooled deionized water to remove excess starch and impurities until the wheat germ is clear in the aqueous solution. Next, thoroughly wash the wheat germ with a 0.5% ethyl phenyl polyethylene glycol solution until the solution is clear and transparent. Grind the wheat germ in a pre-cooled mortar and pestle, adding an equal volume of 1× wheat germ extract and grinding until a dough-like mixture is obtained. Centrifuge the mixture twice at 4 ℃ and 30000g using an ultra-high-speed refrigerated centrifuge for 30 min each time, collecting the supernatant solution between the fat layer and the precipitate. Figure 1 (D). Collect the filtered extract and concentrate the sample. Divide the sample into equal portions and rapidly freeze in liquid nitrogen. Prepare a wheat germ extract with an A260 value of 95-105 for later use.

[0058] Step 3, Fragment Construction: Following the primers in Table 1, construct the expression fragments SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 23 into the form SEQ ID No. 7 + SEQ ID No. 1 / 2 / 3 / 4 / 5 / 23 + SEQ ID No. 8. Mix 25 μL of 2×Taq enzyme, 1 μL of SEQ ID No. 7, 1 μL of SEQ ID No. 1 / 2 / 3 / 4 / 5 / 23, 1 μL of SEQ ID No. 8, and 18 μL of ddH2O. Reaction program: 95 ℃ pre-denaturation for 5 min; 95 ℃ denaturation for 30 s; 60 ℃ annealing for 30 s; 72 ℃ extension for 1.5 min; 72 ℃ final extension for 5 min; after 8 cycles, separate the products by 1% agarose gel electrophoresis.

[0059] Step 4, expression fragment fusion: Add 2 μL of each primer from Table 1 to the reaction solution from the previous step, and perform the following reaction cycle: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s; 60 °C annealing for 30 s; 72 °C extension for 1.5 min; 72 °C final extension for 5 min; after 19 cycles, the product is separated by 1% agarose gel electrophoresis and then purified by gel excision. Figure 2 (A)

[0060] Step 5, in vitro transcription: Mix 10 µL of 5× transcription buffer, 5 µL of nucleoside triphosphate mixture, 5 µL of PCR purified product, 0.5 µL of SP6 RNA polymerase, 0.5 µL of ribonuclease inhibitor, and 29 µL of sterile enzyme-free water.

[0061] Step 6, mRNA purification: Add 10 μL ammonium acetate, 200 μL anhydrous ethanol, and 10 μL sterile enzyme-free water to the reaction solution from the previous step. After incubating on ice for 20 min, centrifuge at 20000 × g and 4 ℃ for 20 min. The product is then separated by 1% agarose gel electrophoresis. Figure 2 (B) The purified mRNA was prepared with sterile water to a concentration of ≥1 μg / μL.

[0062] Step 7, Preparation of cell-free wheat germ system: The purified mRNA obtained in the previous step was mixed with wheat germ extract with A260≈100 and phosphokinase (20 mg / mL) at a volume ratio of 1:1:0.05 and then added to 1× translation supplementation solution. The reaction was carried out at 26 °C for 20 h.

[0063] Step 8: Perform laser confocal microscopy and Western blot analysis on the translation products obtained in Step 7. Figure 3 ).

[0064] Example 3: Preparation of a cell-free system - a two-component hydrogel

[0065] In this embodiment, the multifunctional two-component hydrogel prepared in Example 1 and the wheat germ cell-free protein expression system prepared in Example 2 are mixed to construct a cell-free system-two-component hydrogel. The specific steps are as follows:

[0066] Step 1, Preparation of the two-component hydrogel: The solvent for 5% (w / v) maleimide-based oxidized hyaluronic acid and thiolated carboxymethyl chitosan prepared with deionized water was replaced with 1× translation supplement.

[0067] Step 2, Preparation of expression template: Construct SEQ ID No. 6 into the form SEQ ID No. 7 + SEQ ID No. 6 + SEQ ID No. 8 according to the primers in Table 1. Mix 25 μL of 2×taq enzyme, 1 μL of SEQ ID No. 7, 1 μL of SEQ ID No. 6, 1 μL of SEQ ID No. 8 and 18 μL of ddH2O. The remaining steps are the same as steps 3-6 in Example 2.

[0068] Step 3, Preparation of cell-free system - two-component hydrogel: The mRNA obtained in the previous step was added to the two-component hydrogel prepared with 1× translation supplement solution and reacted at 37 °C for 24 h.

[0069] Step 4: Determination of vascular endothelial growth factor synthesis and release in the cell-free system-two-component hydrogel. Figure 4 ).

[0070] Implementation effect analysis

[0071] The physicochemical properties and performance of the hydrogels prepared in the above embodiments are tested below.

[0072] Test 1: Infrared spectroscopy test.

[0073] Hyaluronic acid, oxidized hyaluronic acid, maleimide-based oxidized hyaluronic acid, carboxymethyl chitosan, thiolated carboxymethyl chitosan, and a two-component hydrogel were used as test samples, with a scanning range of 400 cm⁻¹. -1 –4000 cm -1 The functional group composition and characteristic peaks of each component were observed, and the results are as follows: Figure 5 A.

[0074] Depend on Figure 5 A can be observed at 1730 cm⁻¹. -1 A new characteristic peak appeared nearby, representing the stretching vibration peak of the -C=O bond on the aldehyde group, indicating successful oxidation of hyaluronic acid. Maleimide-oxidized hyaluronic acid showed a peak at 695 cm⁻¹. -1 1650cm -1 1738 cm -1 The appearance of new characteristic peaks at this location, corresponding to the stretching vibrations of -CH, -C=C, and -C=O bonds, indicates the successful preparation of maleimide-based oxidized hyaluronic acid. Thiol-modified carboxymethyl chitosan showed a peak at 606 cm⁻¹. -1 The appearance of a new characteristic peak, representing the stretching vibration peak of the -CS bond, indicates that the preparation of thiolated carboxymethyl chitosan was successful.

[0075] Test 2: Nuclear magnetic resonance hydrogen spectrum test.

[0076] Hyaluronic acid, oxidized hyaluronic acid, maleimide-based oxidized hyaluronic acid, carboxymethyl chitosan, thiolated carboxymethyl chitosan, and a two-component hydrogel were used as test samples. Peak position changes of monomers and polymers were compared at 400 MHz. The results are as follows: Figure 5 B.

[0077] Depend on Figure 5 In Figure B, a strong peak was observed in the chemical shift of oxidized hyaluronic acid near δ 9-10 ppm, representing the stretching vibration peak of the -C=O bond on the aldehyde group, indicating successful oxidation of hyaluronic acid. New characteristic peaks appeared in the chemical shifts of maleimide-oxidized hyaluronic acid at δ 2.5-3.5 ppm and δ 6.5-7.0 ppm, corresponding to the stretching vibration peaks of the -CH and -C=C bonds, respectively, indicating successful preparation of maleimide-oxidized hyaluronic acid. Similarly, a new characteristic peak appeared in the chemical shift of thiolated carboxymethyl chitosan at δ 2.5-3.5 ppm, representing the stretching vibration peak of the -CS bond, indicating successful preparation of thiolated carboxymethyl chitosan. The chemical shifts of the bicomponent hydrogel show new characteristic peaks near δ 2.5-3.5 ppm and δ 8.0-8.5 ppm, which represent -CS and -C=N respectively. This suggests that the gelation mechanism of the bicomponent hydrogel may be a dynamic covalent double crosslinking network formed by the click chemistry reaction of thiol groups and maleimide to generate thioether bonds and Schiff bases formed by aldehyde groups and amino groups.

[0078] Test 3: Differential thermal scanning and thermogravimetric analysis.

[0079] Hyaluronic acid, oxidized hyaluronic acid, maleimide-based oxidized hyaluronic acid, carboxymethyl chitosan, thiolated carboxymethyl chitosan, and a two-component hydrogel were used as test samples. The temperature was increased gradually to 600 °C at a rate of 10 °C / min, with 25 °C as the lower limit. DSC characterization of the samples was performed under a nitrogen atmosphere. The results are as follows: Figure 6 .

[0080] Depend on Figure 6 It can be observed that the thermogravimetric curves of all samples show a downward trend, which is due to the continuous dehydration and weight loss of the samples during the heating process. The total weight loss rates of hyaluronic acid, oxidized hyaluronic acid, and maleimide-based oxidized hyaluronic acid are 68.92%, 69.12%, and 69.79%, respectively; the total weight loss rates of carboxymethyl chitosan and thiolated carboxymethyl chitosan are 56.39% and 57.45%, respectively; and the total weight loss rates of hydrogels with different proportions are between 55.98% and 58.49%, indicating that the network structure can lock in water molecules and maintain its shape.

[0081] Test 4: X-ray diffraction test.

[0082] Hyaluronic acid, oxidized hyaluronic acid, maleimide-based oxidized hyaluronic acid, carboxymethyl chitosan, thiolated carboxymethyl chitosan, and a two-component hydrogel were used as test samples. X-ray diffraction characterization of the samples was performed using Cu single-bond Kα radiation, with a voltage of 40 kV, a current of 40 mA, a scanning range of 2θ = 10–60°, and a scanning frequency of 10 ° / min. The results are as follows: Figure 7 .

[0083] Depend on Figure 7 It can be observed that the peaks of hyaluronic acid, oxidized hyaluronic acid, and maleimide-oxidized hyaluronic acid are all large and weak, indicating the absence of a crystalline structure. Carboxymethyl chitosan exhibits a sharp peak at 2θ = 20°, similar to previous reports, while thiolized carboxymethyl chitosan does not show this peak, indicating that the introduction of thiol groups disrupts the crystalline structure of carboxymethyl chitosan. Conversely, in the hydrogel group, as the proportion of maleimide-oxidized hyaluronic acid increases, new sharp peaks appear near 2θ = 30° and 40°. This indicates that the polymer forms a new interpenetrating network structure through cross-linking, and the increase in the concentration of maleimide-oxidized hyaluronic acid may lead to polymer chain contraction, resulting in a locally ordered structure in the hydrogel, manifested as weak diffraction peaks.

[0084] Test 5: Scanning electron microscopy test.

[0085] The two-component hydrogel was adhered to a silicon substrate as the test sample, and a 5 nm gold / platinum layer was sputtered (10 mA sputtering current, 60 s) to avoid charge accumulation. The hydrogel was observed at 250x magnification in a low-current mode of 5–15 kV. The results are as follows: Figure 8 .

[0086] Depend on Figure 8 It was observed that with the increase of the ratio of maleimide-oxidized hyaluronic acid to thiolated carboxymethyl chitosan, the internal porosity of the gel gradually increased, while the pore size showed a trend of first increasing and then decreasing. When the ratio of maleimide-oxidized hyaluronic acid to thiolated carboxymethyl chitosan was 1:4.5, the internal pore size of the gel was approximately 47 μm; while when the ratio was adjusted to 1:3, the pore size expanded to approximately 55 μm. These results indicate that the ratio of maleimide-oxidized hyaluronic acid to thiolated carboxymethyl chitosan has a significant impact on the porosity and pore size of the hydrogel.

[0087] Test 6: Rheological test.

[0088] The storage modulus and loss modulus of the cured gel were recorded using a flat plate mode. The sol-gel transition was defined as the intersection of the loss modulus / storage modulus curves. Based on oscillatory strain correlation experiments, with a frequency of 1 Hz and a shear strain range of 1%–100% at 25 ℃, frequency correlation experiments were conducted at 25 ℃ with a strain rate of 1%. Dynamic rheological experiments were performed with strains ranging from 1 to 40 rad / s at a frequency of 1 Hz. The results are as follows: Figure 9 .

[0089] Depend on Figure 9 It can be observed that the storage modulus of all hydrogels decreases with increasing shear strain, indicating that their elastic modulus decreases with increasing shear strain. Furthermore, when the shear strain is <10%, the storage modulus of all hydrogel proportions is greater than the loss modulus, indicating that the hydrogels are all elastic solids. When the shear stress is >20%, the 1:2 proportion hydrogel begins to crack; when the shear stress is >46%, the 1:2.5 proportion hydrogel begins to crack; while the remaining hydrogels only begin to crack after the shear stress is >100%, exhibiting good mechanical properties.

[0090] Test 7: Adhesion test.

[0091] A two-component hydrogel was formed in situ on a piece of pigskin after removing surface grease. After gelation and stabilization, forces in different directions (stretching, bending, compression, and torsion) were applied to the skin to observe the adhesion between the hydrogel and the skin. Furthermore, gel stability and tissue adhesion at different pH values ​​(2, 7.4, 10) were measured, and the results are as follows: Figure 10 .

[0092] Depend on Figure 10 It was observed that the hydrogel exhibited poor adhesion in an acidic environment (pH=1.5), and due to the low pH, partial degradation of the gel occurred, resulting in less release of the encapsulated methyl orange dye. In contrast, the hydrogel showed strong adhesion in alkaline environments (pH=10.0 and pH=7.4), with significant dye release observed in the hydrogel at pH=7.4 within 24 hours. After 72 hours, the hydrogel in the acidic environment showed lower adhesion than the hydrogels in the neutral and alkaline environments.

[0093] Test 8: Antibacterial test.

[0094] 1 × 10 6 CFU / mL bacterial suspension ( S. aureus , P. aeruginosa and E. coliThe cells were incubated with PBS and gel for 24 h, respectively. The optical density at 600 nm was measured at fixed time intervals during this period at 37°C. The bacterial suspensions at different treatment times were stained with Calcein AM / Pl Double Staining Kit to distinguish between live and dead cells. After staining, the bacterial suspensions were photographed using an LSM 900. Results are as follows: Figure 11 .

[0095] Depend on Figure 11 It was observed that the hydrogels all exhibited varying degrees of inhibitory effects after co-culturing with bacteria. With prolonged culture time, the optical density values ​​of the hydrogels at each ratio gradually increased, but compared to the control group, all hydrogels at each ratio showed a significant decrease. S. aureus , P. aeruginosa and E. coli The survival rates of the bacteria with the highest inhibition rates at the growth densities were 20.43%, 20.65%, and 4.96%, respectively.

[0096] Test 10: Cytotoxicity and live / dead staining test.

[0097] The cell concentrations of human umbilical vein endothelial cells and human fibroblasts were adjusted to 6 × 10⁻⁶. 3 Cells were evenly seeded into 96-well plates and incubated at 37 ℃ in a 5% CO2 incubator for 24 h. Different groups of hydrogels (Gel), cell-free protein synthesis systems (CF), gel plus cell-free protein synthesis systems (Gel+CF), and gel plus cell-free protein systems expressing vascular endothelial growth factor (Gel+VEGF) were immersed in complete culture medium to obtain extracts. The extracts were then used to replace the basal culture medium in the well plates. After 24 h of incubation, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt was added and incubated for another 3 h. The absorbance was measured at 450 nm using a microplate reader. Similarly, actin-tracing red-rhodamine and Hoechst 33342 staining were used to observe cell morphology, and Calcein AM / Pl DoubleStaining Kit was used to observe live and dead cells. The results are as follows: Figure 12 .

[0098] Depend on Figure 12 It can be observed that both Huvec and Hsf cells effectively adhered to the culture dish, with no significant difference in cell adhesion rate. This indicates that the hydrogel components have minimal impact on cell cytotoxicity. Live / dead staining confirmed the low cytotoxicity of all experimental groups and showed that cell viability was comparable in the test materials.

[0099] Test 11: Cell proliferation test.

[0100] The cell concentrations of human umbilical vein endothelial cells and human fibroblasts were adjusted to 2 × 10⁻⁶. 4 Cells were evenly seeded per well in a 24-well plate and cultured at 37 °C in a 5% CO2 incubator for 24 h. After cell adhesion, the basal medium was replaced with different concentrations of gel extract, and an equal volume of 5-ethyl-2′-deoxyuridine was added to each well. After 3 h of incubation, cells were fixed with 4% paraformaldehyde for 15 min, then cultured with Triton 100 shaking for 10 min, and then treated with EdU click solution for 30 min. The cell nuclei were stained with Hoechst 3342. The results are shown below. Figure 13 .

[0101] Depend on Figure 13 Compared with the PBS control group, enhanced Huvec proliferation was observed in all treatment groups. Quantitative analysis showed that the Gel+VEGF group exhibited the greatest enhancement in proliferation. Although the efficacy of the CF group was lower than that of the hydrogel formulation, all treatment groups were significantly superior to the PBS control group, and the multifunctional hydrogel stimulated proliferation in both Huvec and Hsf.

[0102] Test 12: Cell migration test.

[0103] The cell concentrations of human umbilical vein endothelial cells and human fibroblasts were adjusted to 2 × 10⁻⁶. 4 Cells were evenly seeded per well in a 24-well plate and incubated at 37 °C with 5% CO2 for 24 h. A straight line was drawn at the bottom of each well using a 200 μL pipette tip, followed by rinsing with PBS. The extract was replaced with the basal medium, and after a certain incubation period, the area of ​​the scratch was photographed under a microscope. The results are shown below. Figure 14 .

[0104] Depend on Figure 14 Accelerated migration of Huvecs was observed after 6 hours of incubation. Compared with the PBS control group, Gel, Gel+CF, and Gel+VEGF significantly promoted wound healing, with the Gel+VEGF group showing the greatest efficacy. The reduced effect in the Gel+CF group may reflect the decreased OM / CS content and limited exposure time, and all hydrogel ratios promoted migration.

[0105] Test 13: Infected wound healing test.

[0106] A tissue injury model was established in 70 mice using a 6 mm biopsy wound punch. After model establishment, the mice were randomly divided into four groups: PBS group, cell-free protein synthesis group, 1:3 gel group, 1:3 gel + cell-free protein synthesis group, and 1:3 gel + vascular endothelial growth factor group. 20 μL of Staphylococcus aureus was injected into the wounds of mice in each group to establish a wound infection model. After injection, the wounds were covered with gauze. Mice with successfully established skin infection models underwent different treatments at the wound site every two days to observe healing progress. After 12 days, the mice were sacrificed, and tissue samples from the wounds of each group were excised, fixed with 4% paraformaldehyde, stained with hematoxylin and eosin, and then observed under a microscope to assess the degree of damage. The results are as follows: Figure 15 .

[0107] Depend on Figure 15 A gradual reduction in wound area was observed from day 3 to day 12, with faster wound closure in the Gel+VEGF and Gel+CF groups. By day 12, the wound area reduction was more significant in the Gel+VEGF and Gel+CF groups (99.85±0.06% in the 1:3 gel + vascular endothelial growth factor group and 99.93±0.02% in the 1:3 gel + cell-free protein synthesis group compared to 79.12±1.11% in the PBS group). Histological analysis by H&E staining further confirmed the enhanced epithelialization observed in the Gel+VEGF and Gel+CF groups, with epidermal thickness increasing by 2.34-fold and 2.37-fold, respectively, compared to the PBS group. The Gel+VEGF group showed less inflammatory infiltration accompanied by strong granulation tissue formation, indicating reduced tissue damage caused by infection. Furthermore, the formation of hair follicles and new blood vessels was significantly increased in the CF, Gel+CF and Gel+VEGF groups, further confirming that continuous VEGF synthesis and release promotes the proliferation of endothelial cells and fibroblasts, thereby promoting angiogenesis and improving the wound microenvironment.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for expressing vascular endothelial growth factor using a gel-type wheat germ cell-free protein system, characterized in that, The steps are as follows: (1) Thiol-modified carboxymethyl chitosan and maleimide-oxidized hyaluronic acid were mixed, vortexed, and then allowed to stand to obtain a multifunctional two-component hydrogel; the solvent used for the mixing was 1× translation supplement solution; The maleimide-based oxidized hyaluronic acid is obtained by reacting oxidized hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N-(2-aminoethyl)maleimide under acidic conditions at room temperature with stirring. After the reaction is completed, the mixture is dialyzed with dilute hydrochloric acid solution and lyophilized to obtain maleimide-based oxidized hyaluronic acid. (2) After mixing the template mRNA, wheat germ extract and phosphokinase, add it to the multifunctional two-component hydrogel of step (1) to obtain a gel-type wheat germ cell-free protein system. After reacting at room temperature, vascular endothelial growth factor is obtained. The sequence of the template mRNA is shown as SEQ ID No.7+SEQ ID No.1+SEQ ID No.8, SEQ ID No.7+SEQ ID No.2+SEQ ID No.8, SEQ ID No.7+SEQ ID No.3+SEQ ID No.8, SEQ ID No.7+SEQ ID No.4+SEQ ID No.8, or SEQ ID No.7+SEQ ID No.5+SEQ ID No.8; In step (1), the concentration of thiolated carboxymethyl chitosan is 3-5 wt%; the concentration of maleimide-oxidized hyaluronic acid is 3-5 wt%; the volume ratio of thiolated carboxymethyl chitosan to maleimide-oxidized hyaluronic acid at room temperature is 1:2-1:4.5; the degree of carboxyl substitution of the carboxymethyl chitosan is >80%; the molecular weight of hyaluronic acid is 400,000-800,000; the formulation of the 1× translation supplement is: 120 mM 4-hydroxyethylpiperazine ethanesulfonic acid, 400 mM potassium acetate, 10.8 mM magnesium acetate, 1.6 mM spermidine, 10 mM dithiothreitol, 1.2 mM amino acid standard solution, 4.8 mM adenosine triphosphate, 1 mM guanosine triphosphate, 64 mM creatine phosphate; In step (2), a DNA tag encoding 6 histidine residues was added to the tail of the template mRNA; the volume ratio of template mRNA, wheat germ extract and phosphokinase was 1:1:0.05; the concentration of phosphokinase was 20 mg / mL; the concentration of template mRNA was ≥1 μg / μL; and the A260 value of wheat germ extract was 95-105.

2. The method for expressing vascular endothelial growth factor using a gel-type wheat germ cell-free protein system according to claim 1, characterized in that: In step (1), the molar ratio of oxidized hyaluronic acid monomer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and N-(2-aminoethyl)maleimide is 1:2~3:2~3:2~3; under acidic conditions, the pH is 5~6; and the concentration of the dilute hydrochloric acid solution is 0.01M~0.05M.

3. The method for expressing vascular endothelial growth factor using a gel-type wheat germ cell-free protein system according to any one of claims 1-2, characterized in that, The preparation steps of the thiolated carboxymethyl chitosan are as follows: carboxymethyl chitosan, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and L-cysteine ​​hydrochloride are stirred and reacted at room temperature. After the reaction is completed, the mixture is dialyzed, freeze-dried and then dithiothreitol is added. The mixture is then vacuum filtered to obtain thiolated carboxymethyl chitosan.

4. The method for expressing vascular endothelial growth factor using the gel-type wheat germ cell-free protein system according to claim 3, characterized in that, The molar ratio of chitosan units, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and L-cysteine ​​hydrochloride in the carboxymethyl chitosan is 1:1~2:1~2.

5. A gel-type cell-free wheat germ protein system prepared by the method according to any one of claims 1-2 and 4.

6. The use of the gel-type wheat germ cell-free protein system according to claim 5 in the preparation of functional proteins or products for healing infected wounds.

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