Immune protein sustained-release capsule based on metal polyphenol network as well as preparation method and application of immune protein sustained-release capsule

Abstract

The invention relates to the crossing field of biological materials and agricultural technologies, in particular to an immune protein sustained-release capsule based on a metal polyphenol network as well as a preparation method and application of the immune protein sustained-release capsule. The method comprises the following steps: (1) separating and purifying RxLR23 immune protein, and stabilizing by using a PBS (Phosphate Buffer Solution); (2) preparing a FeCl3 solution and a tannic acid solution; (3) combining the RxLR23 protein with a metal polyphenol network; and (4) dropwise adding the RxLR23-metal polyphenol compound solution into an oil phase, adding a cross-linking agent, stirring, and curing to form the sustained-release capsule. By adopting an emulsification cross-linking method, the effective concentration maintaining time of the immune protein in a plant body is prolonged by 3 times, the lasting period of the immune protein is prolonged, and the utilization rate of foliage spraying is increased. A metal polyphenol network is used as a raw material, the problem that plant immune protein is easy to degrade is solved, meanwhile, tannic acid in the metal polyphenol network has good biocompatibility and oxidation resistance, and the problem that a traditional pesticide sustained-release agent cannot be compatible with protein activity is avoided.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomaterials and agricultural technology, specifically an immune protein sustained-release capsule based on a metal polyphenol network, its preparation method and application. Background Technology

[0002] Metal polyphenol networks (MPNs) are composed of polyphenolic compounds (such as tannic acid, gallic acid, epigallocatechin, etc.) and metal ions (such as Fe). 3+ Cu 2+ Zn 2+ Supramolecular network structures formed through coordination interactions (e.g., polyphenols, etc.) have attracted widespread attention in drug delivery, vaccine development, and immunomodulation due to their unique physicochemical properties and biocompatibility. Metal polyphenol networks show great potential in biomedical applications because they provide a rapid and simple way to construct multifunctional nanoplatforms while exhibiting excellent physicochemical properties and good biocompatibility. Metal polyphenol networks are three-dimensional cross-linked network structures formed by coordination interactions between polyphenolic compounds and metal ions. These materials combine high stability, biocompatibility, and pH responsiveness, showing significant potential in drug delivery, surface coatings, and biosensing.

[0003] The application of immune proteins in agriculture is becoming a key technology for replacing antibiotics and improving the disease resistance of plants and animals. Its core is to achieve green and sustainable disease control by activating the immune system or directly neutralizing pathogens. Plant immune proteins are novel structural proteins isolated and extracted from microorganisms using biotechnology. They can activate the molecular immune and metabolic regulatory systems within plants, improving plant disease resistance while promoting root, stem, and leaf growth and increasing chlorophyll content, thereby increasing crop yield. Currently, existing plant immune protein delivery technologies suffer from problems such as easy degradation, short duration of effectiveness, and low utilization rate when sprayed on leaves. Although metallopolyphenol networks (MPNs) have been studied for drug loading in the pharmaceutical field, there are no reports of their application in agricultural protein slow release. Traditional pesticide slow-release agents are incompatible with protein activity and lack targeted release designs for the soil-plant system. Summary of the Invention

[0004] To address the problems of easy degradation, short duration of effect, and low utilization rate of foliar spraying in plant immune protein delivery technology, and the incompatibility of traditional pesticide slow-release agents with protein activity, this invention provides a method for preparing immune protein slow-release capsules based on a metal polyphenol network.

[0005] On the one hand, the present invention is achieved through the following technical solution: Step 1: Select the immune protein RxLR23 as the core drug component. The RxLR23 immune protein is isolated and purified, and preferably stabilized using PBS buffer at pH 7.4. Step 2: Prepare FeCl3 solution and tannic acid solution, stir, and slowly add tannic acid solution to FeCl3 solution. React for 1-2 hours. The solution turns dark brown and forms a metal polyphenol network solution. Step 3: Slowly add the RxLR23 immunoprotein solution to the formed metal polyphenol network solution and continue stirring for 1-3 hours to promote the binding of RxLR23 immunoprotein to the metal polyphenol network, thus obtaining the RxLR23-metal polyphenol complex solution. Step 4: Using the emulsification crosslinking method, the RxLR23-metal polyphenol complex solution is added dropwise to the oil phase and stirred to form an oil-in-water (W / O) emulsion. Then, a crosslinking agent is added and stirred for 2-4 hours. Microspheres are solidified and formed. After freeze-drying, sustained-release capsules are obtained.

[0006] In a further improvement of the present invention, the immune protein RxLR23 in step 1 is separated and purified by nickel column affinity chromatography to a concentration of 1-3 mg / mL.

[0007] Preferably, the purified concentration of the immune protein RxLR23 is 2 mg / mL.

[0008] In a further improvement of the present invention, the FeCl3 solution prepared in step 2 has a mass concentration of 0.2-0.6 mg / mL, the tannic acid solution has a mass concentration of 0.8-1.2 mg / mL, and the solvent is deionized water.

[0009] Preferably, the FeCl3 solution has a mass concentration of 0.5 mg / mL and the tannic acid solution has a mass concentration of 1 mg / mL.

[0010] In a further improvement of the present invention, the temperature conditions in step 2 are 20-25℃ and the stirring rate is 200-500rpm.

[0011] Preferably, the temperature conditions are 25°C and the stirring speed is 300 rpm.

[0012] In a further improvement of the present invention, in step 2, tannic acid solution is slowly added dropwise to FeCl3 solution, and the molar ratio of FeCl3 solution to tannic acid solution is controlled at 1:1-3.

[0013] Preferably, the molar ratio of FeCl3 solution to tannic acid solution is controlled at 1:2.

[0014] In a further improvement of the present invention, in step 3, the RxLR23 immunoprotein and the metal polyphenol network are bound together under the conditions of pH 5.5-7.5, temperature 4-37℃, and a molar ratio of RxLR23 immunoprotein to metal polyphenol network of 1:1-3.

[0015] Preferably, the RxLR23 immunoprotein and the metal polyphenol network are bound under the conditions of pH 6.5, temperature 25°C, and a molar ratio of RxLR23 protein to metal polyphenol network of 1:2.

[0016] In a further improvement of the present invention, the method used in step 4 is an emulsification crosslinking method, and the added crosslinking agent is one or more of glutaraldehyde, bis(succinimide) octanoate, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the oil phase is corn oil containing Span 80, and the stirring speed is 500-800 rpm.

[0017] Preferably, the crosslinking agent is glutaraldehyde, and the stirring rate is 600 rpm.

[0018] On the other hand, the present invention provides an immunoprotein sustained-release capsule of a metal polyphenol network obtained by the above preparation method.

[0019] Furthermore, this invention provides the application of the immunoprotein sustained-release capsules of the metal polyphenol network obtained by the above method in the prevention and control of plant diseases.

[0020] As can be seen from the above technical solutions, the beneficial effects of the present invention are: This invention utilizes a microsphere-capsule dual-stage sustained-release system prepared by emulsification cross-linking, achieving a cumulative release rate of 88.1% for the immune protein RxLR23 in soil over 30 days. This system extends the effective concentration maintenance time of the immune protein in plants by three times, reduces the incidence of Phytophthora blight by 62.5%, improves the duration of the immune protein's effectiveness, and increases the utilization rate of foliar spraying.

[0021] Metal polyphenol networks (MPNs) are three-dimensional ordered structures formed by the coordination bonds, hydrogen bonds, electrostatic interactions, and van der Waals forces of natural polyphenol molecules. The materials selected are ferric ions (Fe3+). 3+ The metal polyphenol network constructed by combining tannic acid (TA) and metal ions solves the problem of easy degradation of plant immune proteins. The tannic acid in the metal polyphenol network can not only form a stable coordination structure with metal ions, but also has good biocompatibility and antioxidant properties, thus avoiding the problem that traditional pesticide slow-release agents cannot be compatible with protein activity. Attached Figure Description

[0022] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description will be briefly introduced below. Obviously, the accompanying 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.

[0023] Figure 1 The results of RxLR23 immune protein detection in Example 5 of this invention are shown.

[0024] Figure 2 This is to verify the anti-disease effect of RxLR23 immune protein in Example 5 of the present invention. Detailed Implementation

[0025] To make the objectives, features, and advantages of this invention more apparent and understandable, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the embodiments described below are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this patent, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this patent.

[0026] All raw materials used in the examples are commercially available, as follows: ferric chloride, tannic acid, and glutaraldehyde were purchased from Kaitong Chemical Reagent Co., Ltd.; bis(succinimide) octanoate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and PBS buffer were purchased from Sigma-Aldrich. Span 80 corn oil was purchased from Shandong Xingquan Oils Co., Ltd.

[0027] I. Preparation of RxLR23 Immunoprotein Phytophthora capsici is a significant disease affecting pepper production. Researchers have screened RXLR23 through genomic bioinformatics analysis of Phytophthora capsici, obtained the RXLR23 target gene using gene cloning technology, and expressed it in prokaryotic cells of Escherichia coli to obtain highly expressed and high-purity protein. The above process is existing technology. The following is the prokaryotic expression and high-purity protein preparation process of RxLR23 immunoglobulin used in this invention: (a) The constructed RxLR23-pET28a prokaryotic expression vector was transformed into prokaryotic expression Escherichia coli Rossata using the heat shock method.

[0028] (1) Add 2µL of RxLR23-pET28a plasmid to a 1.5ml centrifuge tube, then add 50µL of Rossata competent cells and incubate on ice for 30 min.

[0029] (2) 42℃ water bath for 90 s, followed by ice bath for 2 min.

[0030] (3) Add 500 µL of non-resistant liquid culture medium, incubate at 37°C in a shaker for 45 min, and centrifuge at 6000 rpm for 1 min.

[0031] (4) Discard 300 µL of bacterial supernatant in a clean bench, mix thoroughly by suction and beat, and then drop it onto a plate (with Kana resistance) for plating.

[0032] (5) Incubate at 37℃ upside down for about 12 hours until plaques appear.

[0033] (ii) Perform target protein expression trials and screen for the most suitable expression and purification conditions.

[0034] (1) Add 1 mL of LB (with Kana resistance) to a 2 mL centrifuge tube, pick 12 single spots from the E. coli plate, and incubate in a shaker at 37℃ for 5-6 h.

[0035] (2) After storing 400 µL of Escherichia coli culture, the remaining culture was transferred to new centrifuge tubes and numbered. Control group 1 (CK) was not given IPTG inducer, while the others were given IPTG inducer (concentration of 1 mM / L) in gradients of 0.1-1 µL. Then the tubes were incubated in a shaker at 37℃ for 5 h.

[0036] (3) Centrifuge the E. coli culture at 6000 rpm for 1 min and discard the supernatant. Add 50 µL of 2 × Binding Buffer and resuspend and mix well. Then add 50 µL of 2 × Loading Buffer, mix well, place on a float plate and boil in boiling water for 10 min.

[0037] (4) Perform SDS-PAGE electrophoresis.

[0038] (5) After electrophoresis, the gel was stained with Coomassie Brilliant Blue and then destained. The expression level of the target protein was observed. The bacterial culture with the highest expression level was selected for preservation and subsequent expansion culture.

[0039] (III) Purification of recombinant proteins (1) Add 200 µL of bacterial culture to 20 mL of LB medium (containing Kana), place it in a shaker at 37°C, and incubate for 5-6 h until turbid.

[0040] (2) Add the activated bacterial solution to 1L of sterilized and cooled LB medium to room temperature, and then add 1 mL of Kana resistance.

[0041] (3) In a shaker at 37℃, shake at 180 rpm for 5-6 h until the OD600 value of the culture medium reaches 0.6~0.8.

[0042] (4) When the temperature drops to 16℃, add 1 mL of IPTG (concentration of 1 mM / L). Continue culturing at 120 rpm and 16℃ for 16-20 h.

[0043] (5) Centrifuge the induced bacterial culture at 25°C for 4 min at 8000 rpm, discard the supernatant, and resuspend it in the prepared system solution.

[0044] (6) Use a low-temperature ultra-high pressure continuous flow cell disruptor to disrupt the bacterial resuspension three times at a working pressure of 1032 bar.

[0045] (7) Pre-cool the high-speed refrigerated centrifuge in advance, and centrifuge at 4℃ and 14000 rpm for 25 min.

[0046] (8) Nickel column affinity chromatography: First, equilibrate the affinity chromatography column and fill it with ultrapure water. Pour the supernatant into the column, repeating this process three times. Then, pour the washing buffer into the column and wash three times. Add 20 µL of eluent to the column, plug the end opening of the column, react for 10 min, then open the column. The last eluent is the purified RxLR23 protein. (Note that the protein is easily deformed; store at low temperature.) The FeCl3 solution used in this invention is prepared as follows: Weigh 0.0500 g of anhydrous FeCl3, dissolve it in a small amount of 0.1 mol / L hydrochloric acid, stir until completely dissolved, transfer to a 100 mL volumetric flask, dilute to the mark with deionized water, and shake well.

[0047] II. Preparation of Metal Polyphenol Networks The method for preparing the tannic acid solution used in this invention is as follows: Weigh 100.0 mg of tannic acid powder for later use, take 80 mL of preheated deionized water, add the tannic acid powder, stir magnetically until completely dissolved, transfer to a 100 mL volumetric flask, dilute to the mark with deionized water, and shake well.

[0048] A 0.5 mg / mL FeCl3 solution and a 1 mg / mL tannic acid solution were prepared using deionized water as the solvent. The tannic acid solution was slowly added dropwise to the FeCl3 solution at a stirring rate of 300 rpm at 25 °C, maintaining a molar ratio of 2:1. The reaction was allowed to proceed for 1.5 hours to form a metal polyphenol network.

[0049] The obtained metal polyphenol network was measured, and the results are as follows: (1) Morphological characterization The particle size and distribution of the nanoparticles formed after the reaction were determined by dynamic light scattering. The results showed that pure tannic acid and Fe 3+ The solution has a small particle size (<10 nm). After forming a metal polyphenol network, DLS appears as nanoscale particles with a particle size of about 50-500 nm and a relatively uniform distribution, which is consistent with the characteristics of a nanoscale network structure.

[0050] Table 1 Particle size distribution (2) Chemical characterization Table 2 shows that, according to UV spectrophotometry, the metal polyphenol network solution exhibits a characteristic absorption peak of tannic acid (TA) at ~280 nm, while the Fe³⁺ solution shows weak absorption near ~300 nm, indicating the formation of Fe³⁺. 3+ - After the tannic acid complex is formed, a new absorption peak appears in the 400-600 nm range, indicating that the complexation reaction has occurred.

[0051] Table 2 Absorbance values ​​of each component at different wavelengths III. Solution Synthesis of RxLR23-Metal Polyphenol Complex The protein was purified by nickel column affinity chromatography to a final concentration of 2 mg / mL and stabilized using PBS buffer at pH 7.4. Under different conditions, the RxLR23 immunoprotein solution was slowly added to the pre-formed metallopolyphenol network solution, and stirring was continued for 2 hours to obtain the RxLR23-metallopolyphenol complex solution. The binding rate of RxLR23 immunoprotein to the metallopolyphenol network under different conditions was tested using a physical separation method (taking the sustained-release dispersion and ultracentrifuging (10,000-15,000 rpm, 20 min) to separate the precipitate (bound drug) from the supernatant (free drug)). The results are as follows: Table 3. Binding rates of immunoproteins to metallopolyphenol networks under different conditions The test results showed that the binding rate of the immunoprotein to the metal polyphenol network was highest under the conditions of pH 6.5, temperature 25℃, and a molar ratio of RxLR23 immunoprotein to metal polyphenol network of 1:2.

[0052] The effect of different temperatures on the binding of RxLR23 protein to the metallopolyphenol network was also tested. The stability of the RxLR23 immunoprotein under different temperature conditions with prolonged storage time was determined. The results are as follows: Table 4. Stability of immune proteins under different conditions Test results show that the immune protein exhibits the best stability at 4°C, while maintaining high stability at 25°C.

[0053] IV. Preparation of sustained-release capsules for immune proteins based on metal polyphenol networks Example 1 A method for preparing immune protein sustained-release capsules based on metal polyphenol networks includes the following steps: (1) RxLR23 immunoprotein was separated and purified by nickel column affinity chromatography to a final concentration of 2 mg / mL and stabilized with PBS buffer at pH 7.4.

[0054] (2) Prepare 0.5 mg / mL FeCl3 solution and 1 mg / mL tannic acid solution respectively, using deionized water as solvent. Under 25℃ conditions, slowly add tannic acid solution to FeCl3 solution at a stirring speed of 300 rpm, controlling the molar ratio to be 2:1, and react for 1.5 hours to form a metal polyphenol network.

[0055] (3) Under the conditions of pH 6.5, temperature 25℃, and molar ratio of RxLR23 immunoprotein to metal polyphenol network 1:2, the RxLR23 immunoprotein solution was slowly added to the metal polyphenol network solution that had been formed, and the mixture was stirred for 2 hours to obtain the RxLR23-metal polyphenol complex solution.

[0056] (4) The RxLR23-metal polyphenol complex solution was added dropwise to the oil phase (corn oil containing Span 80) and stirred at 600 rpm to form an oil-in-water (W / O) emulsion. Then glutaraldehyde crosslinking agent was added and stirred for 3 hours to solidify the microspheres.

[0057] The morphology of the microspheres was observed and the particle size distribution was determined using scanning electron microscopy (SEM) and laser particle size analyzer (DLS). SEM observation showed that the microspheres were uniform in morphology, regularly spherical, with smooth surfaces and no obvious breakage or agglomeration. DLS determination showed that the particle size was between 200 and 500 nm, and the PDI (polydispersity index) was less than 0.3, indicating uniform particle size distribution.

[0058] The content of RxLR23 was determined using ultraviolet-visible spectrophotometry (280 nm) and the drug loading and encapsulation efficiency were calculated using the following formula.

[0059] Drug Loading (DL%) = (Mass of RxLR23 in microspheres / Total mass of microspheres) × 100% Encapsulation Efficiency (EE%) = (Mass of RxLR23 in the microspheres / Initial mass of RxLR23 added) × 100% Experimental results showed that the drug loading was 8.5% (w / w) and the encapsulation efficiency reached 91.2%. After freeze-drying, the prepared microspheres had uniform particle size (200~500 nm) and good flowability.

[0060] Using an automated capsule filling machine, dried microspheres are filled into No. 0 gelatin capsule shells to obtain sustained-release capsules.

[0061] Comparative Example 1: RxLR23 immunoprotein was purified by nickel column affinity chromatography and stabilized with PBS buffer. The immunoprotein was applied to the roots of tomato plants, and tomato leaves, stems, and root tissues were collected at different time points using enzyme-linked immunosorbent assay (ELISA). The concentration of RxLR23 immunoprotein was then detected by ELISA to determine its accumulation in the plant. The results showed that the immunoprotein became ineffective within 12-24 hours.

[0062] Comparative Example 2: RxLR23 immunoprotein was purified by nickel column affinity chromatography to a concentration of 2 mg / mL and stabilized with PBS buffer. 0.5 mg / mL FeCl3 and 1 mg / mL tannic acid solutions were prepared using deionized water as the solvent. At room temperature (25°C), the tannic acid solution was slowly added dropwise to the FeCl3 solution at a stirring rate of 300 rpm, maintaining a molar ratio of 2:1, and the reaction was allowed to proceed for 1.5 hours to form a metallopolyphenol network. Under conditions of pH 6.5, temperature 25°C, and a molar ratio of RxLR23 immunoprotein to metallopolyphenol network of 1:2, the RxLR23 immunoprotein solution was slowly added to the formed metallopolyphenol network solution, and stirring was continued for 2 hours to obtain an RxLR23-metallopolyphenol complex solution. This complex solution was uniformly mixed into soil, and release experiments were conducted at 28°C and 50% soil moisture content. The results showed that the release rate of the immunoprotein was too rapid (see Table 5).

[0063] Comparative Example 3: RxLR23 immunoprotein was purified by nickel column affinity chromatography, stabilized with PBS buffer, and the solution was added dropwise to the oil phase. The mixture was stirred to form an oil-in-water emulsion, followed by the addition of a cross-linking agent and stirring for 3 hours to solidify the microspheres. After freeze-drying, sustained-release capsules were obtained. The prepared immunoprotein capsules were applied to the roots of tomato plants. Tomato leaves, stems, and root tissues were collected at different time points (7 days, 14 days, and 21 days) using enzyme-linked immunosorbent assay (ELISA). The concentration of RxLR23 immunoprotein was extracted and detected by ELISA to determine its accumulation in the plant. The results showed that the accumulation of immunoprotein in leaves and stems was relatively low (see Table 6).

[0064] By comparing Example 1 with Comparative Examples 1 and 3, it can be concluded that the present invention solves the problem of easy degradation of immune proteins through the protective effect of MPNs.

[0065] Tannic acid (TA) is rich in catechol / galloyl groups, exhibiting strong antioxidant and metal chelating abilities, and readily binds with Fe. 3+The formed MPNs (Fe-TA) encapsulate / complex the RxLR23 protein, which has the following advantages: It forms a physical barrier, blocking factors in the environment that lead to protein degradation, such as proteases, ultraviolet light, and extreme pH fluctuations.

[0066] By creating an antioxidant microenvironment, TA's strong reducing properties can scavenge reactive oxygen species (ROS) and reduce the damage of oxidative stress to protein activity.

[0067] The phenolic hydroxyl group of TA can form multiple hydrogen bonds and hydrophobic interactions with amino acid residues on the protein surface, stabilizing the native conformation of the protein and preventing it from unfolding and becoming inactive.

[0068] Furthermore, the formation of MPNs and subsequent emulsification and cross-linking are carried out at room temperature and near-neutral pH, minimizing the damage to protein activity caused by harsh conditions such as high temperature, strong acid, and strong alkali.

[0069] By comparing Example 1 and Comparative Example 2, it can be concluded that the present invention solves the problems of short duration of effect and low utilization rate of foliar spraying by using the sustained-release function of microcapsules.

[0070] The microcapsule shell formed by glutaraldehyde cross-linking is the main physical barrier for drug release. The active ingredient (RxLR23-MPN complex) needs to diffuse slowly from the inside of the microcapsule through the polymer network or shell pores to achieve diffusion control.

[0071] Furthermore, the size of microcapsules, the adhesiveness of MPNs, and their sustained-release properties can improve leaf surface retention and absorption. The specific reasons are as follows: The size of the microspheres formed by emulsification and cross-linking helps them adhere to the blade surface, reducing losses caused by rain, wind erosion, or dew rolling off.

[0072] Polyphenols (such as TA) possess excellent adhesion properties, promoting the adhesion of MPNs-protein complexes or microcapsules to leaf surfaces. Fe 3+ It may also interact with components on the blade surface.

[0073] (3) Slow protein release makes the protein concentration reaching the target site more persistent and effective per unit time, avoiding rapid degradation or loss caused by instantaneous exposure to high concentrations of protein.

[0074] This invention solves the problem of traditional pesticide slow-release agents being incompatible with protein activity by binding metal polyphenol networks to immune proteins.

[0075] The protein is first stabilized by MPNs (first layer of protection), followed by TA and Fe. 3+All of these are naturally occurring substances with good biocompatibility. The formation process of MPNs is mild (room temperature, aqueous phase, neutral pH), providing a friendly microenvironment for proteins. The entire MPNs-protein complex is then encapsulated in cross-linked microcapsules (a second layer of protection). This "core-shell" structure maximizes the isolation of proteins from harsh external environments.

[0076] Furthermore, during the emulsification step, glutaraldehyde primarily cross-links with the microcapsule shell material MPNs themselves, interfacially adsorbed molecules, or Span 80 derivatives, forming a physical barrier. Since the RxLR23 protein is already encapsulated by MPNs, harmful cross-linking reactions between glutaraldehyde and the active groups on the protein surface are avoided.

[0077] Therefore, this invention effectively solves the core challenge of plant immune protein delivery through a dual strategy of in-situ mild encapsulation of MPNs and stable emulsification cross-linking microencapsulation. MPNs provide excellent biocompatibility and antioxidant protection, significantly enhancing protein stability; microencapsulation enables controlled sustained release of the drug, greatly extending the duration of action and improving leaf retention capacity; the entire preparation process avoids conditions harmful to protein activity, overcoming the bottleneck of traditional pesticide sustained-release agents being unable to be compatible with the activities of biomolecules.

[0078] Example 2 The difference from Example 1 is that the preparation method of the metal polyphenol network immune protein sustained-release capsule is as follows: (1) RxLR23 immunoprotein was separated and purified by nickel column affinity chromatography to a final concentration of 1 mg / mL and stabilized with PBS buffer at pH 7.4.

[0079] (2) Prepare 0.2 mg / mL FeCl3 solution and 0.8 mg / mL tannic acid solution respectively, using deionized water as solvent. Under 22℃ conditions, slowly add tannic acid solution to FeCl3 solution at a stirring speed of 200 rpm, controlling the molar ratio to 1:1, and react for 1 hour to form a metal polyphenol network.

[0080] (3) Under the conditions of pH 5.5, temperature 37℃, and molar ratio of RxLR23 immunoprotein to metal polyphenol network 1:1, the RxLR23 immunoprotein solution was slowly added to the metal polyphenol network solution that had been formed, and the mixture was stirred for 1 hour to obtain the RxLR23-metal polyphenol complex solution.

[0081] (4) The RxLR23-metal polyphenol complex solution was added dropwise to the oil phase (corn oil containing Span 80) and stirred at 500 rpm to form an oil-in-water (W / O) emulsion. Then, a bis(succinimide) octanoic acid ester crosslinking agent was added and stirred for 2 hours to solidify the microspheres. After freeze-drying, sustained-release capsules were obtained.

[0082] The immunoprotein sustained-release capsules of the metallopolyphenol network were uniformly mixed into the soil, and release experiments were conducted at 28°C and 50% soil moisture content. The results showed that the release of immunoprotein was 20.8% on day 7 and 82.3% on day 21 (see Table 5).

[0083] Example 3 The difference from Example 1 is that the preparation method of the metal polyphenol network immune protein sustained-release capsule is as follows: (1) RxLR23 immunoprotein was separated and purified by nickel column affinity chromatography to a final concentration of 3 mg / mL and stabilized with PBS buffer at pH 7.4.

[0084] (2) Prepare 0.6 mg / mL FeCl3 solution and 1.2 mg / mL tannic acid solution respectively, using deionized water as solvent. Under 20℃ conditions, slowly add tannic acid solution to FeCl3 solution at a stirring speed of 500 rpm, controlling the molar ratio to 1:3, and react for 1.5 hours to form a metal polyphenol network.

[0085] (3) Under the conditions of pH 7.5, temperature 4℃, and molar ratio of RxLR23 immunoprotein to metal polyphenol network 1:3, the RxLR23 immunoprotein solution was slowly added to the metal polyphenol network solution that had been formed, and the mixture was stirred for 3 hours to obtain the RxLR23-metal polyphenol complex solution.

[0086] (4) The RxLR23-metal polyphenol complex solution was added dropwise to the oil phase (corn oil containing Span 80) and stirred at 800 rpm to form an oil-in-water (W / O) emulsion. Then, dimethyl adipate imide crosslinking agent was added and stirred for 4 hours to solidify the microspheres. After freeze-drying, sustained-release capsules were obtained.

[0087] The immunoprotein sustained-release capsules of the metallopolyphenol network were uniformly mixed into the soil, and release experiments were conducted at 28°C and 50% soil moisture content. The results showed that the release of immunoprotein was 21.6% on day 7 and 83.5% on day 21 (see Table 5).

[0088] Example 4 The difference from Example 1 is that the preparation method of the metal polyphenol network immune protein sustained-release capsule is as follows: (1) RxLR23 immunoprotein was separated and purified by nickel column affinity chromatography to a final concentration of 3 mg / mL and stabilized with PBS buffer at pH 7.4.

[0089] (2) Prepare 0.6 mg / mL FeCl3 solution and 1.2 mg / mL tannic acid solution respectively, using deionized water as solvent. Under 25℃ conditions, slowly add tannic acid solution to FeCl3 solution at a stirring speed of 300 rpm, controlling the molar ratio to 1:2, and react for 2 hours to form a metal polyphenol network.

[0090] (3) Under the conditions of pH 6.5, temperature 15℃, and molar ratio of RxLR23 immunoprotein to metal polyphenol network 1:2, the RxLR23 immunoprotein solution was slowly added to the metal polyphenol network solution that had been formed, and the mixture was stirred for 2 hours to obtain the RxLR23-metal polyphenol complex solution.

[0091] (4) The RxLR23-metal polyphenol complex solution was added dropwise to the oil phase (corn oil containing Span 80) and stirred at 800 rpm to form an oil-in-water (W / O) emulsion. Then glutaraldehyde crosslinking agent was added and stirred for 4 hours to solidify the microspheres. After freeze-drying, sustained-release capsules were obtained.

[0092] The slow-release capsules of the metallopolyphenol network were applied to the roots of tomato plants. Tomato leaves, stems, and root tissues were collected at different time points (7 days, 14 days, and 21 days) using enzyme-linked immunosorbent assay (ELISA). After protein extraction, the concentration of RxLR23 immunoprotein was detected by ELISA to determine its accumulation in the plant. The results showed that the application of the slow-release capsules prolonged the effective period of RxLR23 immunoprotein in the plant (see Table 6).

[0093] Example 5: Performance Testing The immunoprotein sustained-release capsules of the metal polyphenol network obtained in Example 1 were tested as follows.

[0094] (1) Land slow-release performance test The immunoprotein sustained-release capsules of the metallopolyphenol network were uniformly mixed in soil, and release experiments were conducted under conditions of 28℃ and 50% soil moisture content. The results showed that the release rate of RxLR23 immunoprotein was low (<20%) in the first 7 days, reached 75% after 21 days, and tended to stabilize after 30 days, indicating that the capsules have a sustained-release effect in soil.

[0095] Table 5. Determination of RxLR23 immunoprotein release rate (2) Plant absorption and bioactivity test Slow-release capsules of a metallopolyphenol network were applied to the roots of tomato plants. Tomato leaves, stems, and roots were collected at different time points (7 days, 14 days, and 21 days) using enzyme-linked immunosorbent assay (ELISA). Proteins were extracted and the concentration of RxLR23 immunoprotein was detected by ELISA to determine its accumulation in the plant. The results showed that the accumulation of RxLR23 immunoprotein in leaves and stems after application of the slow-release capsules was approximately three times higher than that after traditional aqueous solution spraying, and the effective period of RxLR23 immunoprotein in the plant was prolonged.

[0096] Table 6 ELISA Results (ng / g fresh weight) Leaf and stem samples were collected at different time points (7 days, 14 days, and 21 days). RxLR23 immunoprotein was extracted and its presence was detected by Western blot analysis. Figure 1 The figures show the test results for Example 1 and the aqueous solution, respectively.

[0097] (3) Verification of disease resistance effect Fourteen days after applying slow-release capsules or aqueous solutions of a metallopolyphenol network-based immune protein, tomato leaves were artificially inoculated with *Phytophthora infestans*, and disease occurrence was observed. Results showed that the aqueous solution-sprayed group had more severe disease, indicating that the RxLR23 immune protein had a shorter duration of action and had lost its protective effect. In contrast, the plants treated with the slow-release capsules showed a 62.5% reduction in disease incidence, demonstrating its significant role in crop immune regulation. Figure 2 The figures show the test results for Example 1 and the aqueous solution, respectively.

[0098] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an immunoprotein sustained-release capsule based on a metal polyphenol network, characterized in that, Includes the following steps: Step 1: RxLR23 immune protein was isolated and purified, and then stabilized using PBS buffer; Step 2: Prepare FeCl3 solution and tannic acid solution, stir, and slowly add tannic acid solution to FeCl3 solution to form a metal polyphenol network solution; Step 3: Slowly add the RxLR23 immunoprotein solution to the formed metal polyphenol network solution and stir to obtain the RxLR23-metal polyphenol complex solution; Step 4: Add the RxLR23-metal polyphenol complex solution dropwise to the oil phase, stir to form an oil-in-water emulsion, then add a crosslinking agent, stir, and the microspheres solidify. After freeze-drying, sustained-release capsules are obtained.

2. The method for preparing immune protein sustained-release capsules based on metal polyphenol networks according to claim 1, characterized in that: In step 1, the immune protein RxLR23 was separated and purified by nickel column affinity chromatography, with a final concentration of 1-3 mg / mL.

3. The method for preparing immune protein sustained-release capsules based on metal polyphenol networks according to claim 1, characterized in that: In step 2, the FeCl3 solution has a mass concentration of 0.2-0.6 mg / mL, the tannic acid solution has a mass concentration of 0.8-1.2 mg / mL, the solvent is deionized water, and the reaction time is 1-2 hours.

4. The method for preparing immune protein sustained-release capsules based on metal polyphenol networks according to claim 1, characterized in that: In step 2, the temperature conditions are 20-25℃ and the stirring speed is 200-500 rpm.

5. The method for preparing immune protein sustained-release capsules based on metal polyphenol networks according to claim 1, characterized in that: In step 2, tannic acid solution is slowly added dropwise to FeCl3 solution, and the molar ratio of FeCl3 solution to tannic acid solution is controlled at 1:1-3.

6. The method for preparing immune protein sustained-release capsules based on metal polyphenol networks according to claim 1, characterized in that: In step 3, the RxLR23 immunoprotein and the metal polyphenol network bind under the conditions of pH 5.5-7.5, temperature 4-37℃, and a molar ratio of RxLR23 immunoprotein to metal polyphenol network of 1:1-3.

7. The method for preparing immune protein sustained-release capsules based on metal polyphenol networks according to claim 1, characterized in that: The method used in step 4 is emulsification crosslinking. The crosslinking agent added is one or more of glutaraldehyde, bis(succinimide) octanoate, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The oil phase is corn oil containing Span 80, and the stirring speed is 500-800 rpm.

8. An immunoprotein sustained-release capsule of a metal polyphenol network obtained by the method according to any one of claims 1-7.

9. The application of the metal polyphenol network immunoprotein sustained-release capsule obtained by the method according to claim 8 in the prevention and control of plant diseases.

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