A method of extracting gram-positive bacterial outer vesicles

Abstract

This invention belongs to the field of biomedical engineering and discloses a method for extracting exovesicles from Gram-positive bacteria. The method involves culturing Gram-positive bacteria to the logarithmic growth phase to remove residual culture medium components, and then obtaining bacterial exovesicles derived from Gram-positive bacteria through multiple freeze-thaw cycles. These exovesicles contain active substances from the bacteria, such as proteins and small RNA molecules, which can participate in regulating physiological metabolic processes in vivo. Furthermore, they can also serve as drug delivery carriers, crossing membrane systems to target receptor cells via membrane signaling molecules, achieving targeted drug release.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a method for extracting exovesicles of Gram-positive bacteria. Background Technology

[0002] Bacterial extravesicles are nanoscale membrane vesicle structures naturally secreted by bacteria during their growth and metabolism. As an important medium for the exchange of substances and signal transduction between bacteria and their host and microenvironment, they carry bioactive molecules such as bacterial proteins, nucleic acids, and lipids. They can mediate various biological processes such as bacterial virulence transmission, immune regulation, and microbial community interactions. At the same time, due to their natural biocompatibility, targeting, and membrane delivery characteristics, they have become novel nanocarriers for drug delivery, vaccine development, and disease diagnosis in the biomedical field, and have extremely high application value.

[0003] Bacteria can be classified into Gram-positive and Gram-negative bacteria based on Gram staining results. The cell wall structures of these two types differ significantly, directly impacting the release efficiency and extraction difficulty of bacterial exovesicles. Gram-negative bacterial exovesicles can be naturally released through periplasmic accumulation and membrane budding, resulting in relatively high release volumes. In contrast, Gram-positive bacteria cell walls consist of a single, 20-80 nm thick peptidoglycan layer, lacking periplasmic space. This dense peptidoglycan layer forms a natural barrier to exovesicle release, leading to a significantly lower natural release volume compared to Gram-negative bacteria. Currently, there are various extraction methods for exovesicles of Gram-negative bacteria, such as ultracentrifugation, ultrafiltration, and density gradient centrifugation. Each of these methods has its advantages and disadvantages. Traditional ultracentrifugation has low extraction efficiency, requiring long-term centrifugation and washing steps with centrifugal force of over 100,000g. This is not only time-consuming and labor-intensive, but also highly dependent on equipment and prone to causing exovesicle aggregation, membrane structure damage, and loss of their natural biological activity. Although ultrafiltration simplifies the process, it is difficult to effectively separate exoves from bacterial debris, impurities, and other contaminants, resulting in low purity of the extracted product, which cannot meet the needs of subsequent basic research and industrial applications. While density gradient centrifugation can improve purity, it is cumbersome to operate, requires large amounts of reagents, and is prone to low exovesicle recovery rates, increasing extraction costs.

[0004] Gram-positive bacteria, a widely distributed and diverse group of bacteria in nature, encompassing various strains such as Bacillus, Staphylococcus, and Streptococcus, secrete exovesicles that inherit strain-specific bioactivity. These exovesicles not only play a crucial role in the pathogenic mechanisms of pathogenic bacteria, but non-pathogenic Gram-positive bacterial exoves, due to their low toxicity and ease of engineering modification, have become a core research subject for the development of probiotic-derived exovesicles. However, limited by the structural characteristics of Gram-positive bacteria, current exovesicle extraction methods developed for Gram-negative bacteria suffer from numerous insurmountable defects when applied to Gram-positive bacteria, severely restricting basic research and large-scale application of Gram-positive bacterial exovesicles. Therefore, developing an improved extraction method for bacterial exovesicles that is efficient, stable, yield-high, and highly pure, suitable for Gram-positive bacteria, is of great significance for promoting the application of Gram-positive bacterial exoves in the biomedical field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting exovesicles of Gram-positive bacteria. This method is simple, has a significantly higher yield than conventional extraction methods, and produces exovesicles of uniform size, thus preserving their natural conformation and biological activity to the greatest extent.

[0006] A first aspect of the present invention provides a method for extracting exovesicles of Gram-positive bacteria, comprising the following steps:

[0007] S1. Take the Gram-positive bacterial strain that has been stored at low temperature, inoculate it into LB liquid medium under sterile conditions, and place it in a constant temperature shaking incubator for shaking culture until the late logarithmic growth phase;

[0008] S2. After repeated centrifugation of the cultured bacterial solution and resuspending it with PBS buffer, the bacteria are induced to secrete external vesicles through multiple freeze-thaw cycles. The bacterial cells and large particulate impurities are removed by centrifugation and filtration to obtain an external vesicle enrichment solution.

[0009] S3. Extract the outer vesicles from the outer vesicle enrichment solution by polymer precipitation.

[0010] In some embodiments of the present invention, in step S1, the inoculation of the Gram-positive bacterial strain stored at low temperature into LB liquid medium under sterile conditions specifically involves: taking the Gram-positive bacterial strain stored at -80℃, and inoculating 1 mL of the strain into LB liquid medium using a pipette in a sterile laminar flow hood.

[0011] In some embodiments of the present invention, the pH of the LB liquid culture medium is 7.2-7.6.

[0012] In some embodiments of the present invention, the concentration of tryptone in the LB liquid culture medium is 10 g / L, the concentration of yeast extract is 5 g / L, and the concentration of NaCl is 10 g / L.

[0013] In some embodiments of the present invention, the Gram-positive bacterial species is Bacillus subtilis.

[0014] In some embodiments of the present invention, step S1, which involves placing the bacteria in a constant temperature shaking incubator and shaking it until the late logarithmic growth phase, specifically involves shaking it in a constant temperature shaking incubator at a temperature of 37°C and a rotation speed of 180-220 rpm for 12-16 hours until the bacteria grow to the late logarithmic growth phase.

[0015] In some embodiments of the present invention, in step S2, the cultured bacterial solution is washed with pre-cooled PBS buffer to remove residual culture medium components: the cultured bacterial solution is transferred to a centrifuge tube, centrifuged, the supernatant is discarded, and the bacterial cell pellet is collected. Pre-cooled 0.01 mol / L PBS buffer (pH 7.4) is added to the bacterial cell pellet, with a volume of 1 / 10 of the initial bacterial solution volume, and the pellet is gently resuspended by pipetting. The pellet is centrifuged again, and the supernatant is discarded. The washing process is repeated 2-3 times to thoroughly remove residual culture medium components. The preferred centrifugation conditions are: 0-4℃, 2500-3500 rpm for 5-15 min.

[0016] In some embodiments of the present invention, in step S2, the centrifugation speed is 2500-3500 rpm and the centrifugation time is 5-15 min.

[0017] In some embodiments of the present invention, in step S2, the freeze-thaw process specifically involves freezing followed by thawing, with the freezing temperature being -75℃ to 80℃ and the freezing time being 30 to 60 minutes, and the thawing temperature being 35℃ to 37℃ and the thawing time being 5 to 10 minutes.

[0018] In some embodiments of the present invention, in step S2, the number of freeze-thaw cycles is 1-5. More freeze-thaw cycles are not necessarily better. As the number of freeze-thaw cycles continues to increase beyond 5, the extracted outer vesicle membrane structure is easily damaged, and vesicles are prone to fusion, resulting in a significant reduction in the yield of target outer vesicles with a diameter of less than 200 nm.

[0019] In some embodiments of the present invention, in step S2, the pore size of the filter membrane used for filtration is 0.22-0.45 μm.

[0020] In some embodiments of the present invention, in step S3, the polymer precipitation method is as follows: add polyethylene glycol 6000 solution to the vesicle enrichment solution, vortex mix, and incubate at 4°C for 12-16 h; centrifuge at 10000-11000 rpm for 30-60 min, collect the precipitate and resuspend it with PBS buffer to obtain the vesicles.

[0021] The method for extracting exovesicles of Gram-positive bacteria according to embodiments of the present invention has at least one of the following advantages:

[0022] 1. Compared to the passive extraction approach that relies solely on the natural secretion of bacteria in existing technologies, this method uses a gentle freeze-thaw cycle to artificially and controllably disrupt the peptidoglycan barrier, forcibly and efficiently releasing a large number of extravesicles retained intracellularly and intercellularly. This increases the extraction yield of Gram-positive bacterial extravesicles by 4 to 15 times or more, fundamentally solving the problem of dense cell walls and extremely low natural vesicle release in Gram-positive bacteria.

[0023] 2. In the extraction method of this invention, the freeze-thaw and subsequent precipitation processes are under mild conditions, which can effectively avoid mechanical damage to the outer vesicle membrane structure and maintain its natural conformation and biological activity to the greatest extent. As a drug delivery carrier, it can effectively achieve drug enrichment in vivo and has important application value in the fields of tissue damage repair and inflammatory disease treatment. Attached Figure Description

[0024] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of preferred embodiments taken in conjunction with the accompanying drawings, in which:

[0025] Figure 1 This is a transmission electron microscope image of the Bacillus subtilis exovesicles prepared in Example 1;

[0026] Figure 2 This is a line graph comparing the protein concentrations extracted from Bacillus subtilis exovesicles prepared after different freeze-thaw cycles (1, 2, 3, 5, 7, and 10 times). In this graph, the horizontal axis represents the number of freeze-thaw cycles, and the vertical axis represents the protein concentration of the exovesicles (μg / mL). Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention and should not be construed as a limitation of the present invention.

[0028] LB liquid medium: tryptone concentration of 10 g / L, yeast extract concentration of 5 g / L, and NaCl concentration of 10 g / L.

[0029] Example 1

[0030] (1) Take the Bacillus subtilis strain stored at -80℃, and inoculate 1 mL of the strain into LB liquid medium using a pipette in a sterile laminar flow hood. Incubate in a constant temperature shaking incubator at 37℃ and 200 rpm for 16 h until the bacteria reach the late logarithmic growth phase. Transfer the cultured bacterial solution to a centrifuge tube and centrifuge at 4℃ and 3000 rpm for 15 min. Discard the supernatant and collect the bacterial pellet. Add 30 mL of pre-cooled 0.01 mol / L, pH 7.4 PBS buffer to the bacterial pellet, gently resuspend by pipetting, and centrifuge again at 4℃ and 3000 rpm for 15 min. Discard the supernatant and repeat the washing process twice.

[0031] (2) Add 12 mL of 0.01 mol / L, pH 7.4 PBS buffer to the washed bacterial pellet, resuspend, and aliquot into 12 1.5 mL microcentrifuge tubes. Perform freeze-thaw cycles: first freeze at -80℃ for 30 min, then thaw in a 37℃ water bath for 5 min. Centrifuge the freeze-thawed enriched solution at 4℃ and 3000 rpm for 15 min, collect the supernatant, and filter through a 0.22 μm filter membrane to obtain 8 mL of exovesicle enriched solution.

[0032] (3) The enriched solution was subjected to vesicle extraction using a polymer precipitation method. 8 mL of 10% PEG6000 solution was added to the enriched solution, and the mixture was allowed to stand at 4°C for 16 h. Subsequently, the mixture was centrifuged at 10,000 rpm for 30 min at 4°C, the supernatant was discarded, and the precipitate was collected. 5 mL of pre-cooled 0.01 mol / L, pH 7.4 PBS buffer was added to the precipitate for resuspending. The resuspended solution was transferred to an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa), and concentrated to a volume of 1.3 mL by centrifugation at 3000 rpm at 4°C. The purified Bacillus subtilis vesicle solution was obtained. The transmission electron microscopy image of the Bacillus subtilis vesicles is shown below. Figure 1 As shown.

[0033] Example 2

[0034] In this embodiment, the freeze-thaw cycle (freezing in an ultra-low temperature freezer at -80℃ for 30 min and then thawing in a water bath at 37℃ for 5 min) in step (2) is repeated twice. The remaining extraction operation steps and conventional control extraction methods are completely consistent with those in Example 1.

[0035] Example 3

[0036] In this embodiment, the freeze-thaw cycle (freezing in an ultra-low temperature freezer at -80℃ for 30 min and then thawing in a water bath at 37℃ for 5 min) in step (2) is repeated 3 times. The remaining extraction operation steps and conventional control extraction methods are completely consistent with those in Example 1.

[0037] Example 4

[0038] In this embodiment, the freeze-thaw cycle (freezing in an ultra-low temperature freezer at -80℃ for 30 min and then thawing in a water bath at 37℃ for 5 min) in step (2) is repeated 5 times. The remaining extraction operation steps and conventional control extraction methods are completely consistent with those in Example 1.

[0039] Example 5

[0040] In this embodiment, the freeze-thaw cycle (freezing in an ultra-low temperature freezer at -80℃ for 30 min and then thawing in a water bath at 37℃ for 5 min) in step (2) is repeated 7 times. The remaining extraction operation steps and conventional control extraction methods are completely consistent with those in Example 1.

[0041] Example 6

[0042] In this embodiment, the freeze-thaw cycle (freezing in an ultra-low temperature freezer at -80℃ for 30 min and then thawing in a water bath at 37℃ for 5 min) in step (2) is repeated 10 times. The remaining extraction operation steps and conventional control extraction methods are completely consistent with those in Example 1.

[0043] Comparative example (0 freeze-thaw cycles)

[0044] Take Bacillus subtilis culture stored at -80℃ and inoculate 1 mL of the culture into LB broth using a pipette in a sterile laminar flow hood. Incubate at 37℃ and 200 rpm for 16 h with shaking until the bacteria reach the late logarithmic growth phase. Transfer the culture to a centrifuge tube and centrifuge at 4℃ and 3000 rpm for 15 min. Filter the supernatant through a 0.22 μm filter to obtain 90 mL of pre-extract.

[0045] The pre-extract was subjected to exovesicle extraction using a polymer precipitation method. 90 mL of 10% (w / v) PEG6000 solution was added to the pre-extract, and the mixture was incubated at 4°C for 16 h. Subsequently, the mixture was centrifuged at 10,000 rpm for 30 min at 4°C, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in 5 mL of pre-cooled 0.01 mol / L, pH 7.4 PBS buffer. The resuspended solution was transferred to an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa), and concentrated to a volume of 1.1 mL by centrifugation at 3,000 rpm at 4°C, yielding a purified Bacillus subtilis exovesicle solution obtained using conventional methods.

[0046] The extravesicle suspensions obtained in Examples 1-6 and the comparative example were used as test samples. Extravesicle proteins were extracted from each suspension. The total protein solution obtained was then quantitatively analyzed using the BCA method to characterize the relative protein concentration of the corresponding extravesicle. The results are as follows: Figure 2 As shown in the figure, the line trend indicates that when the number of freeze-thaw cycles is between 1 and 5, the concentration of outer vesicle proteins increases with the increase in the number of freeze-thaw cycles. When the number of freeze-thaw cycles exceeds 5, the protein concentration shows a significant decreasing trend, which intuitively reflects that the optimal operating range for freeze-thaw cycles is 1-5. When the number of freeze-thaw cycles exceeds 5, the concentration of outer vesicle proteins shows a decreasing trend. Since the extracted proteins come from outer vesicles with a diameter of less than 200 nm in the sample, the decrease in protein concentration intuitively reflects a significant reduction in the number of source outer vesicles.

Claims

1. A method for extracting exovesicles of Gram-positive bacteria, characterized in that, The method includes the following steps: S1. Take the Gram-positive bacterial strain preserved at low temperature, inoculate it into LB liquid medium under aseptic conditions, and place it in a constant temperature shaking incubator to shake and culture until the late logarithmic growth phase to obtain the cultured bacterial solution; S2. Wash the cultured bacterial solution with pre-cooled PBS buffer to remove residual culture medium components, then induce the bacteria to secrete exovesicles through multiple freeze-thaw cycles, and remove bacterial cells and large particulate impurities by centrifugation and filtration to obtain an exovesicle enrichment solution. S3. Extract the outer vesicles from the outer vesicle enrichment solution by polymer precipitation.

2. The method as described in claim 1, characterized in that, In step S1, the pH of the LB liquid medium is 7.2-7.

6.

3. The method as described in claim 1, characterized in that, In step S1, the concentration of tryptone in LB liquid medium is 10 g / L, the concentration of yeast extract is 5 g / L, and the concentration of NaCl is 10 g / L.

4. The method as described in claim 1, characterized in that, The Gram-positive bacterial species is Bacillus subtilis.

5. The method according to claim 1, characterized in that, In step S1, the shaking culture in a constant temperature shaking incubator specifically means: shaking culture in a constant temperature shaking incubator at a temperature of 37℃ and a rotation speed of 180-220 rpm for 12-16 hours until the bacteria grow to the late logarithmic growth phase.

6. The method according to claim 1, characterized in that, In step S2, the centrifugation speed is 2500-3500 rpm and the centrifugation time is 5-15 min.

7. The method according to claim 1, characterized in that, In step S2, the freeze-thaw process specifically involves freezing first and then thawing, with the freezing temperature being -75℃ to 80℃ and the freezing time being 30 to 60 minutes, and the thawing temperature being 35℃ to 37℃ and the thawing time being 5 to 10 minutes.

8. The method according to claim 1, characterized in that, In step S2, the number of freeze-thaw cycles is 1-5.

9. The method according to claim 1, characterized in that, In step S2, the pore size of the filter membrane used for filtration is 0.22-0.45 μm.

10. The method according to claim 1, characterized in that, In step S3, the polymer precipitation method is as follows: add polyethylene glycol 6000 solution to the outer vesicle enrichment solution, vortex mix, and incubate at 4°C for 12-16 h; centrifuge at 10000-11000 rpm for 30-60 min, collect the precipitate and resuspend it with PBS buffer to obtain the outer vesicle.

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