CN115074301A - Method for enriching bacterial extracellular vesicles by using epsilon-polylysine - Google Patents

Abstract

The invention relates to the field of biological medicine, in particular to a method for enriching bacterial extracellular vesicles by using epsilon-polylysine. The invention provides a method for enriching bacterial extracellular vesicles from a biological sample by using epsilon-polylysine for the first time. Compared with the classical ultracentrifugation method, the method only needs a common high-speed centrifuge and does not need large expensive centrifugal equipment; compared with the methods of tangential flow filtration, still water filtration and the like, the method does not need to design and manufacture a special filtering device, and is simple and convenient to operate; compared with ultrafiltration and affinity separation methods, the method has unique large-volume sample processing capacity by adopting a precipitation mode; the reagent used in the invention comprises epsilon-polylysine, has lower cost compared with the antibody and the aptamer used in the affinity separation method, and has better economic practicability and clinical application prospect. In addition, the method has the potential of precipitating bacterial vesicles from complex samples such as body fluid and the like, and can promote the development of bacterial vesicle detection and application research.

Description

Method for enriching bacterial extracellular vesicles by using epsilon-polylysine

Technical Field

The invention relates to the field of biological medicine, in particular to a method for enriching bacterial extracellular vesicles by using epsilon-polylysine.

Background

Bacterial Extracellular Vesicles (BEV) are a type of membrane vesicles produced by bacteria and secreted extracellularly, typically between 20-200 nm in diameter. Secretory extracellular vesicles are conserved functions that exist in a wide variety of bacteria including gram-positive and gram-negative bacteria. BEV is encapsulated by a lipid bilayer, with the outer bacterial vesicle membrane being homologous to the outer bacterial cell membrane for gram-negative bacteria, and having the outer cell membrane component Lipopolysaccharides (LPS) on its surface, and being homologous to the cell membrane for gram-positive bacteria, and also having a portion of the bacterial cell wall component, such as lipoteichoic acid (LTA), on its surface. BEV contains a variety of biomolecules, such as proteins, nucleic acids, lipids, etc., the contents of which can be released into the surrounding environment or into other cells to perform different functions. BEV plays a diverse role in the growth, proliferation and communication of information between cells of bacteria. BEV is widely existed in human hosts, is an important medium for communication between bacteria and hosts, and has great application potential in diagnosis and treatment. Because the BEV has tiny particle size, the BEV is difficult to separate from a biological sample, and brings difficulty to the development of the field.

Epsilon-polylysine is a natural broad-spectrum cationic antimicrobial peptide produced by fermentation with actinomycetes. The outer vesicle surface of the bacterial cell is rich in negative charges and can interact with epsilon-polylysine with positive charges.

The most common BEV extraction method at present is density-based Ultracentrifugation (UC): bacterial cells and most of the cell debris etc. were first removed by high speed centrifugation and 0.22 μm or 0.45 μm filters, then the supernatant was centrifuged for a minimum of 2h with an ultracentrifugation of more than one hundred thousand g, the pellet was collected and subsequently BEV purified by ultracentrifugation again with one-step washing of the resuspension.

In addition, there are separation methods based on the size of the BEV, such as Ultrafiltration (UF), shear flow filtration (TFF) and Hydrostatic Filtration (HF). For the ultrafiltration method, the bacterial cell depleted supernatant was ultracentrifuged through a 100-500kDa ultrafiltration tube. Shear flow filtration is a relatively new method that also uses a filter membrane, except that the direction of the filter membrane and the direction of the liquid flow are arranged tangentially, to some extent reducing the clogging of the filter membrane by the BEV. Still water filtration the supernatant containing BEV was filtered by applying hydrostatic pressure to a 1000kDa filter, followed by resuspension of the concentrated supernatant, and filtration through a 0.45 μm filter, again purification by a second ultrafiltration step, and static dialysis, and finally concentrated collection through filter tubes.

The affinity-based separation methods include mainly an antibody method and a nucleic acid aptamer method. Both methods are based on some characteristic proteins on the surface of the BEV, antibodies or aptamers which are specifically bound are designed and connected with carriers which are easy to enrich, such as histone labels or magnetic beads, and after the antibodies or aptamers are bound with the BEV, the BEV is collected by enriching the labels or magnetic beads which are bound with the antibodies or aptamers.

Previous techniques have not focused on isolating bacterial extracellular vesicles, which belong to prokaryotes, whose cell membranes differ greatly in composition and in relation to mammalian membrane systems, resulting in vesicles that are also significantly different in nature from mammalian extracellular vesicles. For example: cholesterol is an important component of mammalian exosomes, while bacterial membrane systems typically do not contain cholesterol; bacterial extracellular vesicles generally contain bacterial specific components such as lipopolysaccharides and lipoteichoic acids, which mammalian extracellular vesicles are unlikely to contain. Thus, bacterial extracellular vesicles and mammalian exosomes cannot be considered as the same entity, and researchers are essentially different from previous technologies in the target.

The most common existing ultracentrifugation method consumes long time, needs trained technicians to operate, is expensive in equipment and consumables, and is difficult to process samples with large volumes; ultrafiltration and other separation methods based on size are easy to cause the blockage of a filter membrane, increase the treatment time, and some methods also need to design special filter devices; although the affinity-based method has better specificity, the selection of the universal marker on the surface of the BEV still needs to be researched, so that part of BEV without the specific marker can be lost by the affinity-based enrichment method, the affinity-based method has certain disadvantages in terms of processing sample volume and processing efficiency, vesicles of bacterial origin cannot be precipitated from a complex sample, the precipitation capacity of epsilon-polylysine on the bacterial extracellular vesicles is stronger than that of mammalian extracellular exosomes, the potential of selectively separating the bacterial extracellular vesicles from a mixed sample is realized, and the method is obviously different from the previous method in terms of target and effect.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for enriching bacterial extracellular vesicles by using epsilon-polylysine, so as to improve the separation efficiency of bacterial extracellular vesicles. The invention provides a method for enriching bacterial extracellular vesicles from a biological sample by using epsilon-polylysine for the first time.

Epsilon-polylysine is a natural broad-spectrum cationic antimicrobial peptide produced by fermentation with actinomycetes. The outer vesicle surface of the bacterial cell is rich in negative charges and can interact with epsilon-polylysine with positive charges.

The invention provides a method for separating bacterial outer vesicles, which comprises the steps of incubating epsilon-polylysine and the bacterial outer vesicles, centrifugally collecting precipitates, and replacing the solution after washing, releasing and ultrafiltration to obtain the vesicles. Compared with the existing classical ultracentrifugation separation method, the method only needs to use a common high-speed centrifuge (about 10000 g) in the aspect of instruments, does not need to use expensive large ultracentrifugation equipment, and greatly improves the practicability of the method; compared with the methods of tangential flow filtration, still water filtration and the like, the method does not need to design and manufacture a special filtering device, is simple and convenient to operate, and can be operated by ordinary experimenters through simple learning.

In the method, before the co-incubation, MES buffer solution is mixed with bacterial liquid, and the pH value is adjusted to 7; the volume ratio of the MES buffer solution to the bacterial liquid is 1: 9.

In the method, the concentration of the epsilon-polylysine in the co-incubation step is 30-100 mu g/mL; in some embodiments, the co-incubation is at a concentration of epsilon-polylysine of 5, 15, 30, 60, or 100 mu g/mL; in some embodiments, the concentration of epsilon-polylysine in the co-incubation step is 100. mu.g/mL.

In the method, the co-incubation time is 15-60 min, and the room temperature is 18-30 ℃; in some embodiments, the co-incubation time is 15min, 30 min, 45min, or 60 min; in some embodiments, the co-incubation time is 45 min.

In the method, the centrifugation conditions comprise a rotation speed of 2,000-12,000 g at 4 ℃ and a time of 4-16 min; in some embodiments, the conditions of the centrifugation comprise a 4 ℃ spin rate of 500g, 2,000g, 5000g, 6000g, 8000g, 9000g, or 12,000g for 4min, 8min, 12 min, or 16 min;

in some embodiments, the incubated mixture is centrifuged at 12,000g for 15min at 4 ℃.

In the method of the present invention, the washing comprises washing 2 times with a PBS solution. In some embodiments, the washing comprises centrifuging and discarding the supernatant, resuspending the pellet in 1mL PBS, centrifuging at 12,000g for 15min at 4 deg.C, and repeating this process once more. Because the method adopts a precipitation mode, compared with an ultrafiltration and affinity separation method, the method has unique large-volume sample processing capacity.

In the method, the releasing comprises the step of resuspending the precipitate by using a release buffer solution, wherein the release buffer solution is 50 mM Tris, 0.5M-1M NaCl, and the pH value is 8-9. In some embodiments, the releasing comprises resuspending the resulting pellet with 500. mu.L of release buffer at room temperature 18 ℃ to 30 ℃ for 5 min.

In the method of the present invention, the ultrafiltration comprises a concentration multiple of 5 times of the volume of the liquid after ultrafiltration; the specification of the ultrafiltration tube is 100 kDa.

In the method of the present invention, the ultrafiltration is to separate epsilon-polylysine from bacterial extracellular vesicles.

In some embodiments, the solubilized solution is centrifuged briefly to remove insoluble precipitates, the supernatant is added to a 100kDa ultrafiltration tube, centrifuged at 14,000 g at 4 ℃ until the volume of the solution in the tube is reduced to 1/5, supplemented with release buffer to the initial volume, centrifuged until the volume of the solution in the tube is reduced to 1/5 again, and the process is repeated 3 times.

In the method, the release buffer solution comprises 50 mM Tris and 0.5-1M NaCl, and the pH value is 8-9.

In the method of the invention, the displacement solution comprises displacement with a PBS solution until the filtrate pH is 7. In some embodiments, the permutation comprises: the ultrafiltrate was added to 4 volumes of PBS buffer at 4 ℃ to the ultrafiltration tube, and the mixture was centrifuged at 14,000 g at 4 ℃ until the volume of the solution in the ultrafiltration tube was reduced to 1/5, and the pH of the filtrate was measured using pH paper. The procedure of adding PBS buffer solution and centrifuging is repeated until the pH value of the filtrate is restored to 7; bacterial outer vesicles were obtained.

In the method of the present invention, the bacterium includes a gram-positive bacterium and/or a gram-negative bacterium. In the present invention, the separation effect of the method on gram-negative bacterial vesicles is verified by taking escherichia coli as an example, and the separation effect of gram-positive bacterial vesicles is verified by taking staphylococcus aureus as an example.

The invention also provides the vesicle prepared by the method.

The provided vesicles are bacterial outer vesicles, which are enriched by the method of the invention.

The invention provides a method for enriching bacterial extracellular vesicles from a biological sample by using epsilon-polylysine for the first time. Compared with the existing classical ultracentrifugation separation method, the method only needs to use a common high-speed centrifuge (about 10000 g) in the aspect of instruments, does not need to use expensive large ultracentrifugation equipment, and greatly improves the practicability of the method; compared with methods such as tangential flow filtration and still water filtration, the method does not need to design and manufacture a special filtration device, is simple and convenient to operate, and can be operated by ordinary experimenters through simple learning; because the method adopts a precipitation mode, compared with an ultrafiltration and affinity separation method, the method has unique large-volume sample processing capacity; in addition, the present invention is essentially different from the previous technology in the aspect of the extracted object, the present invention has the potential of selectively separating extracellular vesicles derived from bacteria from a mixed sample, and epsilon-polylysine is added into diluted fetal calf serum or a cell culture medium, so that protein cannot be separated under the conditions of the method, but bacteria-related protein can be precipitated when the bacterial vesicles are mixed in serum.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts according to the drawings:

FIG. 1 shows a schematic diagram of the process of enrichment of outer vesicles of bacteria by epsilon-polylysine, wherein the enrichment process comprises four steps of incubation and precipitation of epsilon-polylysine, centrifugal collection and precipitation, heavy suspension of the precipitate and ultrafiltration of a displacement solution;

FIG. 2 illustrates morphological validation of BEV; FIGS. A-D are transmission electron micrographs of BEV, and A in FIG. 2 is E.coli BEV obtained by ultracentrifugation; b in FIG. 2 is E.coli BEV obtained by the epsilon-polylysine precipitation method; FIG. 2C shows the enrichment of Staphylococcus aureus BEV by ultracentrifugation; d in FIG. 2 is the Staphylococcus aureus BEV obtained by the epsilon-polylysine precipitation method, and it can be seen that this method and the classical ultracentrifugation method can obtain similar BEV with the vesicle type of the saucer shape; E-H is BEV particle size distribution obtained by nano-dynamics tracking; e in FIG. 2 is E of E.coli BEV enriched by ultracentrifugation; FIG. 2F is E.coli BEV obtained by the epsilon-polylysine precipitation method; FIG. 2G shows the enrichment of Staphylococcus aureus BEV by ultracentrifugation; FIG. 2 is a diagram showing the distribution of the particle size of Staphylococcus aureus BEV obtained by the epsilon-polylysine precipitation method, which shows that the distribution of the particle size of BEV obtained by the present method is substantially the same as that obtained by the ultracentrifugation method;

FIG. 3 shows the isolated BEV biological functional assay; FIG. 3A shows the cytotoxicity tests of E.coli and S.aureus BEVs obtained by the two methods, four BEVs showing no significant cytotoxicity to human monocyte THP-1 cells during the treatment time; B-D in FIG. 3 are mRNA expression levels of three immune factors IL-1 beta (B), IL-6(C) and IL-8(D) after BEV stimulation of THP-1 cells, and it can be seen that four BEV stimulations can all cause the significant increase of the immune factor expression level compared with a negative control; E-F in FIG. 3 is the protein secretion level of two immune factors IL-8 (E) and IL-1 beta (F) after BEV stimulation of THP-1 cells, and it can be seen that compared with the control, the secretion of the immune factors can be obviously improved by four BEV stimulations; in the figure, EUC represents Escherichia coli BEV obtained by ultracentrifugation, EPL represents Escherichia coli BEV obtained by epsilon-polylysine precipitation, SUC represents Staphylococcus aureus BEV obtained by ultracentrifugation, and SPL represents Staphylococcus aureus BEV obtained by epsilon-polylysine precipitation;

FIG. 4 shows the protein gel electrophoresis of BEV obtained by precipitation under different concentrations of epsilon-polylysine, which shows that epsilon-polylysine of 30. mu.g/mL or more can effectively precipitate BEV;

FIG. 5 shows protein gel electrophoresis of BEV precipitated at different incubation times, which shows that incubation for 45min or more effectively precipitated BEV;

FIG. 6 shows protein gel electrophoresis images of BEV obtained at different centrifugation rotation speeds, which shows that the BEV can be effectively precipitated at 2,000-12,000 g rotation speed;

FIG. 7 shows protein gel electrophoresis of BEV obtained at different centrifugation times, which shows that centrifugation at 8min or more is effective in obtaining BEV precipitate;

FIG. 8 shows a protein gel electrophoresis diagram with 9 lanes from left to right, wherein lanes 1, 4 and 7 show the protein size marker; 2. lane 3 is the result of the isolation of exosomes from a mammalian cell line, lane 2 is the result of ultracentrifugation, and lane 3 is the result of the isolation according to the present invention; no bands were evident in lane 3, indicating that epsilon-polylysine was unable to isolate mammalian exosomes; 5. lanes 6 and 7, 8 show the results of E.coli and S.aureus separations, respectively, and it can be seen that bands similar to those obtained by ultracentrifugation can be obtained using the method of the present invention;

FIG. 9 shows the separation of epsilon-polylysine and one of the antimicrobial peptides polymyxin B; although polymyxin B is a cationic antimicrobial peptide with epsilon-polylysine, polymyxin B lacks separation ability under the same conditions for BEV of gram-negative bacteria escherichia coli and BEV of gram-positive bacteria staphylococcus aureus; the sedimentation of BEV by the epsilon-polylysine is not only related to the interaction of positive and negative charges of the BEV, but also can depend on other special properties of the epsilon-polylysine;

FIG. 10 shows that extracellular vesicles of E.coli can be efficiently precipitated at a centrifugation speed of 2000g or more;

FIG. 11 shows that vesicles can be efficiently separated with centrifugation times of more than 8 minutes;

FIG. 12 shows that vesicles can be efficiently separated by adding 15. mu.g or more of epsilon-polylysine per ml of the medium;

FIG. 13 shows that addition of epsilon-polylysine to diluted fetal bovine serum or cell culture medium does not allow for separation of proteins under the conditions of the process, but allows for precipitation of bacterially related proteins when bacterial vesicles are incorporated in the serum.

Detailed Description

The invention provides a method for enriching bacterial extracellular vesicles by using epsilon-polylysine, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The method specifically comprises the following steps:

1. aggregation and separation of epsilon-polylysine and bacterial extracellular vesicles

Under the condition of pH 6-7, 30-100 mu g/mL of epsilon-polylysine is incubated to agglomerate the bacterial extracellular vesicles, so that the separation of the bacterial extracellular vesicles is realized at the centrifugal speed of 2000-12000 g.

2. Separation of epsilon-polylysine from bacterial extracellular vesicles

(1): under the conditions that the pH value is 8-9 and the NaCl concentration is 0.5-1M, epsilon-polylysine can fall off from the surface of the bacterial extracellular vesicle, so that the aggregate is recovered into a single vesicle.

(2): the ultrafiltration can separate vesicles with molecular mass of more than 100kDa from epsilon-polylysine with low relative molecular mass (the relative molecular mass is 3600 Da-4300 Da), and the solution is replaced to disperse the vesicles in PBS buffer solution, so that the method can be directly used for detecting components and functions.

Preparing solution required by experiment

MES buffer: 3M MES, 0.15M NaCl, pH 5 adjusted with NaOH

Releasing buffer solution: 50 mM Tris, 0.5M NaCl, pH 8.5 with HCl

PBS buffer: pH 7.4

Bacterial culture and pretreatment of culture media

Inoculating the bacteria into LB culture medium, and shaking culture at 37 deg.C overnight;

centrifuging the culture medium at room temperature with 6000g for 10 min, discarding precipitate, centrifuging the supernatant at 4 deg.C and 10000g for 15min, collecting supernatant, and sterilizing with 0.45 μm filter.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

EXAMPLE 1 Epsilon-polylysine precipitation of BEV

200mL of the bacterial culture medium after centrifugal filtration pretreatment is taken, the pH of the bacterial culture medium is detected by using a wide pH test paper, and if the alkalinity is too much deviated, MES buffer solution is taken and added into the pretreated bacterial culture medium in a ratio of 1:9, and the pH value of the culture medium is adjusted to 7.

Weighing 100mg of epsilon-polylysine, preparing 10 mg/mL of mother liquor by using PBS buffer solution, adding the mother liquor into 200mL of bacterial culture medium with the pH value adjusted to 7 until the final concentration of the epsilon-polylysine is 100 mu g/mL, wherein the example results of different concentrations of the epsilon-polylysine are shown in figure 4;

placing the bacterial culture medium added with the epsilon-polylysine on a rocker bed of a rocker, uniformly mixing and incubating for 45min at room temperature, wherein the results of the examples with different incubation times are shown in figure 5;

the incubated mixture was centrifuged at 12,000g for 15min at 4 ℃ and the results of the examples at different centrifugation speeds and centrifugation times are shown in FIGS. 6 and 7, respectively, the supernatant was carefully discarded, the pellet was resuspended in 1mL PBS, centrifuged at 12,000g for 15min at 4 ℃ and this procedure was repeated once more, and the resulting pellet was lysed with 500. mu.L of release resuspension buffer at room temperature for 5 min;

centrifuging the dissolved solution for a short time to remove insoluble precipitate, adding the supernatant into a 0.5 mL 100kD ultrafiltration tube, centrifuging at 4 deg.C and 14,000 g until the volume of the solution in the ultrafiltration tube is reduced to about 100 μ L, adding release buffer to 0.5 mL, centrifuging until the volume of the solution in the ultrafiltration tube is reduced to about 100 μ L, and repeating the process for 3 times.

PBS buffer was added to the tube to 0.5 mL, centrifuged at 14,000 g at 4 ℃ until the volume of the solution in the tube was reduced to about 100. mu.L, and the pH of the filtrate was measured using pH paper. The procedure of adding PBS buffer solution and centrifuging is repeated until the pH value of the filtrate is restored to 7;

the 200. mu.L of liquid in the ultrafiltration tube was removed for subsequent analysis and detection.

Example 2 Experimental parameters affecting the results

The separation conditions of various extracellular vesicles, such as centrifugation time, concentration, centrifugation speed, and the like, were tested using a medium in which escherichia coli was cultured, and the amount of protein obtained was detected using a protein gel electrophoresis method. A protein band around 35 kD is used as Escherichia coli, and the band contains various Escherichia coli outer membrane proteins such as OmpA, OmpC and OmpF, and is a well-known Escherichia coli extracellular vesicle marker.

2.1 the conditions except the centrifugation speed are the same as those in example 1, and the effect of the centrifugation speed on the extraction effect is studied, and the results show that the extracellular vesicles of Escherichia coli with the centrifugation speed of 500 g-12000 g can be effectively precipitated at the centrifugation speed of more than 2000g, as shown in FIG. 10.

2.2 the conditions except the centrifugation time are the same as those in example 1, and the influence of the centrifugation time on the extraction effect is studied, and the results show that the centrifugation time is 4-16 minutes, and the vesicles can be effectively separated when the centrifugation time exceeds 8 minutes, as shown in fig. 11.

2.3 in addition to the concentration of epsilon-polylysine, the same conditions as in example 1 were used to examine the effect of the concentration of epsilon-polylysine on the extraction, and the results showed that the concentration of epsilon-polylysine, when 15. mu.g or more of epsilon-polylysine was added per ml of the medium, was effective in separating vesicles, as shown in FIG. 12.

Comparative example 1 Ultracentrifugation (UC) separation of BEV

200mL of the bacterial culture medium after the pretreatment of centrifugal filtration was taken, centrifuged at 4 ℃ for 2h using 160,000g, the supernatant was carefully discarded, then the pellet was resuspended in 1mL of PBS solution, centrifuged again at 160,000g for 2h, the supernatant was discarded, and the pellet was resuspended in 100. mu.L of PBS for subsequent analysis and detection.

1. Transmission Electron Microscopy (TEM) for identifying BEV morphology

Diluting BEV by a proper multiple, dropwise adding 7 mu L of suspension on a 300-mesh copper net, incubating at room temperature for 2min, carefully sucking the supernatant from the side by using filter paper, dropwise adding 7 mu L of uranium acetate, incubating at room temperature for 1min, carefully sucking the supernatant from the side by using filter paper, completely drying the copper net, and observing the form of the bacterial extracellular vesicles under a transmission electron microscope.

2. NTA identification of BEV particle size distribution

The resulting BEV suspension was diluted 1000-fold with distilled water and the BEV particle size distribution was measured with a Nanoparticle Tracking Analyzer (NTA).

3. BEV biological function identification

And (5) verifying the effect of BEV and immune cells. THP-1 mononuclear cells are treated by PBS (phosphate buffered saline) solution with certain concentration of BEV for 6h, and the mRNA expression level of immune factors such as IL-8, IL-6, IL-1 beta and the like is detected by fluorescent quantitative PCR (qPCR). The secretion of the immune factor in the cell culture supernatant after stimulation was examined by enzyme-linked immunosorbent assay (ELISA).

Comparative example 2 antimicrobial peptide polymyxin B precipitate BEV

Bacterial BEVs were isolated with the antimicrobial peptide polymyxin B according to the protocol of example 1, and the results are shown in figure 9. It can be seen that although polymyxin B is a cationic antimicrobial peptide with epsilon-polylysine. However, polymyxin B lacks the ability to isolate the BEV of the gram-negative bacterium Escherichia coli and the BEV of the gram-positive bacterium Staphylococcus aureus under the same conditions. Indicating that the precipitation of BEV by epsilon-polylysine is not only related to its positive and negative charge interactions, but may also depend on other characteristic properties of epsilon-polylysine. Epsilon-polylysine is non-toxic, inexpensive, and an ideal substance for the treatment of large volume samples, as shown in fig. 9.

Effect verification

1. The epsilon-polylysine adopted by the invention has certain selectivity aiming at bacterial extracellular vesicles and mammal exosomes. As shown in FIG. 8, the protein was stained blue by the Coomassie brilliant blue stain. There are 9 lanes from left to right in the figure, of which lanes 1, 4 and 7 are markers indicating the size of the protein. 2. Lane 3 shows the result of the separation of exosomes of the mammalian cell line by the method of example 1, lane 2 shows the result of the separation by ultracentrifugation of comparative example 1, and lane 3 shows the result of the separation by the method of example 1; it can be seen that there is no distinct band in lane 3, indicating that epsilon-polylysine is unable to separate mammalian exosomes. 5. Lanes 6 and 7, 8 show the results of E.coli and S.aureus separations, respectively, and it can be seen that the method of example 1 produces bands similar to those obtained by ultracentrifugation. Therefore, epsilon-polylysine has a stronger precipitating ability to bacterial extracellular vesicles than mammalian extracellular exosomes, has the potential to selectively separate bacterial extracellular vesicles from a mixed sample, and is significantly different from the method of comparative example 1 in both target and effect.

2. The method of example 1 has the potential to selectively isolate bacterial-derived extracellular vesicles in a mixed sample. As shown in FIG. 13, addition of epsilon-polylysine to diluted fetal bovine serum or cell culture medium failed to isolate protein under the conditions of the present method. When bacterial vesicles are mixed in the serum, bacteria-associated proteins can be precipitated, as shown in fig. 13. Therefore, the method in example 1 can precipitate bacteria-derived vesicles from complex samples such as body fluids, and can promote the development of bacterial vesicle detection and application research.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A method for separating bacterial outer vesicles is characterized by comprising the steps of incubating epsilon-polylysine and the bacterial outer vesicles, centrifuging, collecting precipitates, and replacing the solution after washing, releasing and ultrafiltering to obtain the vesicles.

2. The method of claim 1, further comprising mixing a MES buffer with the bacterial suspension prior to the co-incubation, adjusting the pH to 7; the volume ratio of the MES buffer solution to the bacterial liquid is 1: 9.

3. The method according to claim 1 or 2, wherein the concentration of epsilon-polylysine in the co-incubation step is between 30 and 100 μ g/mL.

4. The method according to any one of claims 1 to 3, wherein the co-incubation time is 15min to 60min at a room temperature of 18 ℃ to 30 ℃.

5. The method of claim 1, wherein the centrifugation conditions comprise a 4 ℃ rotation speed of 2,000g to 12,000g for 4min to 16 min.

6. The method of claim 1, wherein the washing comprises washing 2 times with a PBS solution; the releasing comprises using a release buffer solution to resuspend the sediment, wherein the release buffer solution is 50 mM Tris, 0.5M-1M NaCl, and the pH value is 8-9.

7. The method of claim 1, wherein the ultrafiltration comprises a 5-fold concentration of the post-ultrafiltration liquid volume; the specification of an ultrafiltration tube adopted by ultrafiltration is 100 kDa.

8. The method of claim 1, wherein the displacing solution comprises displacing with a PBS solution until the filtrate has a pH of 7.

9. The method of claim 1, wherein the bacteria comprise escherichia coli and/or staphylococcus aureus.

10. A vesicle prepared by the method of any one of claims 1 to 9.

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