CN-121022721-B - Purification method of plant exosomes

Abstract

The invention discloses a purification method of plant exosomes, which belongs to the technical field of biological separation, and comprises the steps of obtaining plant juice or tissue extract, carrying out rough filtration and centrifugal pretreatment, incubating the plant juice or tissue extract with magnetic microspheres with carboxyl or epoxy groups modified on the surfaces in a buffer solution containing calcium ions, capturing a compound by using an external magnetic field, eluting by adopting a competitive elution buffer solution containing N-acetylglucosamine after washing, and finally further purifying by a size exclusion chromatographic column to obtain the plant exosomes. The method provided by the invention has the advantages that the exosomes are specifically captured by the magnetic microspheres, and the recovery rate and purity of the exosomes are obviously improved by combining the double purification strategies of competitive elution and size exclusion chromatography, so that the method is simple and convenient to operate, short in time consumption and good in repeatability, and is suitable for large-scale preparation of exosomes of various plant sources.

Inventors

• ZHANG HAIXIN
• LI HUAWEI

Assignees

• 京美生命科技(杭州)有限公司

Dates

Publication Date

20260512

Application Date

20251029

Claims (2)

1. 1. A method for purifying tomato leaf exosomes, comprising the steps of: obtaining a tomato leaf tissue extract, and removing large-particle impurities through rough filtration and centrifugation to obtain a pretreatment liquid; mixing and incubating the pretreatment liquid and the modified magnetic microspheres under a buffer condition; After the incubation is completed, an external magnetic field is applied to capture the magnetic microsphere combined with the exosome of the tomato leaf, and buffer solution is adopted for cleaning to obtain a magnetic microsphere-exosome compound; Adding competitive eluting buffer solution containing N-acetylglucosamine into the magnetic microsphere-exosome compound, and incubating to obtain eluent; Immediately loading the obtained eluent to a pre-balanced size exclusion chromatographic column, and collecting target liquid to obtain high-purity tomato leaf exosomes; The modified magnetic microsphere is a polystyrene magnetic microsphere with a carboxyl-rich surface, and the particle size of the polystyrene magnetic microsphere is 1-3 mu m; the concentration of N-acetylglucosamine in the competitive elution buffer is 5-20wt%; The filler of the size exclusion chromatographic column is porous silica microsphere with the surface modified by polyethylene glycol, the pore diameter is 30-50nm, the particle diameter is 5-10 mu m, the molecular weight of the polyethylene glycol with the surface modified by the filler is 2000-5000Da, and the modification density is 0.5-1.2mg/m < 2 >; the competitive elution buffer also contains 0.1-0.5wt% chitosan oligosaccharide; The modified magnetic microsphere is subjected to pre-activation treatment before incubation, and the pre-activation solution is MES buffer solution containing 0.5-1.5wt% of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride; The pre-activation treatment time is 20-40 minutes, and the temperature is 20-25 ℃; The buffer condition is phosphate buffer solution containing 0.1wt% to 0.5wt% CaCl 2 ; the pretreatment liquid is added with 0.05-0.2wt% of polyethylene glycol octyl phenyl ether.
2. 2. The purification method according to claim 1, wherein the mass-to-volume ratio of the modified magnetic microsphere to the pretreatment liquid is 1g (10-50) mL, the incubation time is 30-60min, and the incubation temperature is 4-25 ℃.

Description

Purification method of plant exosomes Technical Field The invention belongs to the technical field of biological separation, and particularly relates to a purification method of plant exosomes. Background Exosomes are nanoscale vesicles actively secreted by cells, are widely existing in animal and plant body fluids, have lipid bilayer membrane structures, carry bioactive molecules such as proteins, nucleic acids, lipids and the like, and have important functions in the aspects of intercellular communication, immunoregulation, disease diagnosis, treatment and the like. In recent years, plant exosomes are becoming research hotspots in the fields of drug delivery, functional foods, skin care products, product development in respiratory health and sensory health, and the like, due to the advantages of wide sources, high biocompatibility, low immunogenicity, and the like. However, efficient purification of plant exosomes remains a difficulty in the current technology. The traditional ultracentrifugation method is widely applied, but has the problems of complex operation, long time consumption, easy aggregation or damage of exosomes and the like, the polymer precipitation method is simple, convenient and quick, has more impurity residues, seriously affects the reliability and safety of downstream application, and has the advantages that the size exclusion chromatography, ultrafiltration and other technologies improve the purity to a certain extent, but still can not realize the unification of high recovery rate and high specificity. In addition, plant samples often contain a large amount of interfering substances such as polysaccharide, phenols, pigments and the like, so that the difficulty of exosome separation is further increased. Therefore, developing a method for separating plant exosomes with high efficiency, high speed and high purity has important scientific research and industrial value. In view of this, the present invention has been made. Disclosure of Invention The first object of the present invention is to provide a purification method of plant exosomes, which combines magnetic microspheres to specifically capture exosomes and a double purification strategy of competitive elution and size exclusion chromatography, so that the recovery rate and purity of exosomes are remarkably improved, the operation is simple and convenient, the time consumption is short, the repeatability is good, the method is suitable for large-scale preparation of exosomes from various plant sources, and the method has wide application prospects in the fields of drug delivery systems, cosmetics, product development in respiratory health and sensory health, etc. In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: A method for purifying plant exosomes, comprising the steps of: obtaining plant juice or plant tissue extract, and removing large-particle impurities through rough filtration and centrifugation to obtain pretreatment liquid; mixing and incubating the pretreatment liquid and the modified magnetic microspheres under a buffer condition; After the incubation is completed, an external magnetic field is applied to capture the magnetic microsphere combined with the plant exosome, and buffer solution is adopted for cleaning to obtain a magnetic microsphere-exosome compound; Adding competitive eluting buffer solution containing N-acetylglucosamine into the magnetic microsphere-exosome compound, and incubating to obtain eluent; And immediately loading the obtained eluent to a pre-balanced size exclusion chromatographic column, and collecting target liquid to obtain the high-purity plant exosomes. The invention provides a purification method of plant exosomes, which is characterized in that a high-efficiency, specific and mild separation and purification platform is constructed by the technical scheme, firstly, complex plant raw materials are preprocessed to remove macroscopic impurities, a foundation is laid for subsequent fine operation, then, the specific modified magnetic microspheres are used as a capturing medium, high-efficiency and selective adsorption of the target exosomes is realized under the assistance of an external buffer condition, then, solid-liquid rapid separation is realized by applying an external magnetic field, the operation efficiency is greatly improved, the loss of the target substances is reduced, then, an elution strategy based on a competitive displacement principle is adopted, the complete exosomes are gently released from a solid-phase carrier, the damage of vesicle structures or the loss of biological activity possibly caused by severe chemical or physical conditions is effectively avoided, finally, any trace impurities possibly remained are further removed, a fine purification step of size exclusion chromatography is introduced, and finally, a high-purity plant exosomes product is obtained, the method is deeply analyzed, firstly