CN106811428A - It is a kind of to extract the method that membrane vesicle is arranged outside bacterium - Google Patents

Abstract

The invention discloses a method for obtaining outer membrane vesicles of Escherichia coli and Klebsiella pneumoniae by using an ultrafiltration concentration method. Cultivate overnight in a test tube containing a medium, then inoculate an appropriate amount of bacterial solution into a shaker flask containing 1 medium, and cultivate the OD value close to 1.0, collect the bacterial solution, centrifuge to remove the bacterial cells, filter through a sterile filter head to remove the residual bacterial cells, Collect the filtrate, centrifuge and concentrate to 1/8 of the original volume with an ultrafiltration tube with a molecular weight cut-off of 100KDa, add physiological saline to wash and repeat 2 to 3 times, collect the bacterial outer membrane vesicles on the upper layer of the ultrafiltration tube, and store at -20°C The environment was subpackaged and frozen, and the concentration of the extracted Klebsiella pneumoniae outer membrane vesicle protein was 1.5 mg/ml, and that of Escherichia coli was 2.1 mg/ml, and the particle size of OMVs was about 20-100 nm. The method is convenient and simple to operate, and provides a new method for the extraction and research of OMVs.

Description

A method for extracting bacterial efflux membrane vesicles

technical field

The invention relates to a method for preparing escherichia coli (DH5α) and Klebsiella pneumoniae (klebsiella pneumoniae, KP) efflux membrane vesicles (bacterial outer membrane vesicles, OMVs) by using an ultrafiltration concentration method.

Background technique

Bacterial outer membrane vesicles are reported to be bilayered spherical proteoliposomes that are continuously released in the form of budding from the surface of Gram-negative bacteria, generally 20–250 nm in diameter, and usually carry bacterial outer membrane and periplasmic components, including Some DNA, RNA, endotoxin (LPS), protein, enzyme and peptidoglycan, etc., their structures are shown in Figure 1; clinical studies have shown that OMVs can cause inflammation and pathological responses in the body, and OMVs are associated with many infectious diseases , such as periodontitis, gastritis, Crohn's disease, salpingitis, meningitis, sepsis, cardiovascular disease, and lung disease, OMVs have attracted more and more attention from researchers.

Studies have shown that OMVs themselves have some inherent characteristics, such as the ability to produce immunogenic surface and membrane-associated components similar to those of the mother, and can enhance the response of antibodies and T cells to antigens, and are often used to make vaccines; experiments have been carried out It has been shown that it is very stable under different temperature and treatment conditions, and it is a non-replicating vaccine. It has been reported that OMVs obtained through genetic engineering modification can selectively express corresponding antigens, reduce endotoxicity, and are widely used.

In recent years, biological nanocarriers derived from bacteria, viruses, mammalian cells, etc. have received extensive attention and research as drug delivery systems. Drugs (such as chemotherapy drugs and siRNA, etc.) to specific target sites and other advantages, OMVs are also widely used as such a biological nanocarrier in the study of drug delivery (Kaparakis-Liaskos M, Ferrero RL (2015) Immune modulation by bacterialouter Membrane vesicles. Nature Reviews Immunology 15(6):375-387 doi:10.1038/nri3837. Schwechheimer C, Kuehn MJ (2015) Outer-membranevesicles from Gram-negative bacteria: biogenesis and functions. Nature Reviews Microbiology 13(10) : 9doi :10.1038/nrmicro3525.).

The current extraction methods for OMVs mainly include sucrose density gradient centrifugation and Optiprep density gradient centrifugation. However, practice shows that the two methods are cumbersome to operate and troublesome in post-processing. In view of this, the inventors of the present application intend to provide a convenient and simple method for extracting bacterial efflux membrane vesicles (OMVs).

Contents of the invention

The purpose of the present invention is to overcome the defects and deficiencies of the prior art, and provide a method for extracting bacterial efflux membrane vesicles (OMVs) which is easy to operate and simple to implement.

The present invention utilizes the ultrafiltration concentration method, and on this basis, adds physiological saline to wash the OMV _S , removes impurities such as redundant miscellaneous proteins and flagella pili, and obtains a relatively pure extract. Compared with the density gradient centrifugation method, the method of the present invention is convenient to operate, Simple and easy to implement, it provides a new method for the extraction and research of OMVs.

The method for extracting bacterial efflux membrane vesicles (OMVs) of the present invention uses the ultrafiltration concentration method to obtain Escherichia coli and Klebsiella pneumoniae outer membrane vesicles, mainly comprising: making Escherichia coli and Klebsiella pneumoniae bacteria Suspension, take an appropriate amount of bacterial suspension and inoculate it in a test tube containing medium for overnight cultivation, then take an appropriate amount of bacterial suspension and inoculate it in a shaker flask containing medium to cultivate an OD value close to 1.0, collect the bacterial solution, and centrifuge to remove the bacteria, aseptic Filter the filter head to remove residual bacteria, collect the filtrate, centrifuge and concentrate to 1/8 of the original volume with an ultrafiltration tube with a molecular weight cut-off of 100KDa, add normal saline to wash and repeat this step for 2 to 3 times, and collect in the upper layer of the ultrafiltration tube Bacterial outer membrane vesicles were aliquoted and stored at -20°C.

In the present invention, the Escherichia coli DH5α is from the American Culture Collection, and the Klebsiella pneumoniae is from the China Agricultural Microorganism Collection.

More specifically, the method for extracting bacterial efflux membrane vesicles (OMVs) of the present invention comprises:

First, two strains of Gram-negative bacteria, Klebsiella pneumoniae and Escherichia coli DH5α, were selected and cultured in NB medium and LB medium respectively until the OD value reached 1.0, and the bacterial cells were removed by high-speed centrifugation, and then 0.22 μM no Thoroughly sterilize the bacteria by membrane filtration, transfer the culture medium to the ultrafiltration concentrator tube to concentrate to 1/8 of the original volume, wash 2 to 3 times with normal saline to obtain relatively pure OMVs, observe the morphology of OMVs under a transmission electron microscope, SDS - PAGD gel electrophoresis to observe the protein amount and molecular weight contained in OMVs; BCA method to measure the concentration of outer membrane vesicle protein of Klebsiella pneumoniae extracted is about 1.5mg/ml, and the concentration of outer membrane vesicle protein of Escherichia coli is about 2.1mg /ml, the particle size of OMVs observed under a transmission electron microscope is about 20-100nm.

In the present invention, the OMVs are double-layered star proteoliposomes continuously released from the surface of Escherichia coli and Klebsiella pneumoniae in the form of budding, usually carrying bacterial outer membrane and periplasmic components.

In the present invention, Escherichia coli and Klebsiella pneumoniae are made into bacterial suspension, first get 10 μ L of bacterial suspension in 3ml culture medium (Escherichia coli is cultivated in LB medium, and Klebsiella pneumoniae is cultivated in NB medium ) cultured overnight for 12 hours, and transferred the test tube bacterial solution to corresponding 40ml culture medium for culture until the OD value reached 1.0.

In the present invention, the bacterial solution is placed in a 50ml centrifuge tube, centrifuged at 6000r/min for 20min, the bacterial precipitate is discarded to obtain the culture supernatant, and then filtered with a 0.22 μM sterile filter head to obtain a sterile filtrate.

In the present invention, the obtained sterile filtrate is added to a 50ml ultrafiltration tube, centrifuged at 3000r/min, the OMVs are trapped in the upper part of the ultrafiltration tube, and the centrifugation is repeated until the total volume is concentrated to 1/8 of the original volume, about 5ml, OMVs were obtained after cleaning.

In the present invention, the cleaning method includes adding 5 ml of normal saline (0.9% NaCl solution) to the OMVs obtained by sucrose density gradient centrifugation, transferring them to ultrafiltration tubes for ultrafiltration and concentration, and repeating 2-3 times to obtain pure The OMV.

The bacterial outer membrane vesicles obtained in the present invention can be further used as a pharmaceutically acceptable nano drug delivery carrier. and further used in the preparation of vaccines.

The present invention uses the ultrafiltration concentration method to obtain the outer membrane vesicles of Escherichia coli and Klebsiella pneumoniae, can obtain a relatively pure extract, and the concentration of the extracted Klebsiella pneumoniae outer membrane vesicle protein is about 1.5mg/ml , the outer membrane vesicle protein concentration of Escherichia coli is about 2.1mg/ml, and the particle size of OMVs observed under a transmission electron microscope is about 20-100nm; compared with the prior art density gradient centrifugation method, it is more convenient and simple to operate, and is OMVs The extraction and related research work provide a new method.

Description of drawings

Figure 1. Schematic diagram of Klebsiella pneumoniae.

Figure 2, Klebsiella pneumoniae (left) and Escherichia coli colonies (right).

Figure 3, Morphology of Klebsiella pneumoniae (left) and Escherichia coli (right) under an inverted microscope.

Figure 4. Protein-absorbance standard curve.

Figure 5 Electron micrographs of OMVs of Klebsiella pneumoniae (left) and Escherichia coli (right).

Fig. 6 SDS-PAGE electrophoresis image, wherein, the left image is Klebsiella pneumoniae OMVs, and the right image is Escherichia coli OMVs.

detailed description:

Embodiment 1: Selection and cultivation of bacterial strains

Klebsiella pneumoniae was purchased from the China Agricultural Microorganism Culture Collection, and Escherichia coli DH5α was purchased from the American Culture Collection. Klebsiella pneumoniae was cultured with NB medium, and Escherichia coli was cultured with LB medium; the freeze-dried powder of the strains was dissolved in sterile saline, and an appropriate amount was placed on a solid medium, and cultured overnight at 37°C until a single colony (as shown in the figure) 2); single colonies were cultured in a test tube containing 3ml of liquid medium, and cultured overnight at 37°C and 180r/min on a shaker. The next day, 3ml of the bacterial liquid was transferred to a shake flask containing 40ml of medium for culture until the OD value of the bacteria reached 1.0. Figure 3 shows the morphology of the bacteria after staining with crystal violet.

Embodiment 2: Extraction of OMVs

Centrifuge the bacterial solution cultivated to an OD value of 1.0 in a 50ml sterile centrifuge tube at 6000r/min for 20min to remove most of the bacterial cells, filter the supernatant with a 0.22μM sterile filter head to completely sterilize, and then transfer the filtrate Centrifuge at 3000r/min for 10min at 4°C for 10min in 50ml of ultrafiltration tube with a molecular weight cut-off of 100KD, and repeat several times until the overall concentration of the solution is about 1/8 of the original;

Add 5ml of normal saline to the concentrated solution, use the ultrafiltration concentration method to wash away impurities such as excess protein and flagella pili, dispense it into EP tubes, and store at -20°C.

Example 3: Identification of OMVs

A, BCA method to the determination of OMVs protein concentration;

OMVs protein concentration was measured by BCA method;

Take 5 mg/ml standard protein in a 96-well plate to establish a protein-absorbance standard curve (as shown in Figure 4), take 2 μl of OMVs samples in the other 5 secondary wells, add 200 μl of AB solution to each well (A solution: B solution = 50:1), incubate in an incubator at 37°C for 30 minutes, measure the absorbance value of each well at a wavelength of 570nm; establish a standard curve for protein content, R ² =0.9989>0.99, showing a good linear relationship;

The measured protein concentration of Klebsiella pneumoniae OMVs is about 1.5 mg/ml, and the concentration of Escherichia coli OMVs is about 2.1 mg/ml;

Table 1 shows the protein concentration of the extracted Klebsiella pneumoniae and Escherichia coli OMVs.

Table 1

B. Negative stain electron microscopy to detect the morphology of OMVs

The OMVs of the two extracted Gram-negative bacteria were dropped onto the carbon-coated copper grid, 2% uranyl acetate was added dropwise, dried at room temperature, and sent to the electron microscope room of the Analysis and Testing Center of East China University of Science and Technology in Shanghai for observation. The results showed that, The particle size of OMVs of Klebsiella pneumoniae and Escherichia coli is about 20-100nm, and the morphology is shown in Figure 5;

C. SDS-PAGE observation of OMVs protein types

The results of 15% SDS-PAGE electrophoresis showed that the two extracted OMVs contained multiple protein bands. Klebsiella pneumoniae OMVs mainly contained complex proteins with molecular weights of 60KDa and 110KDa, while Escherichia coli mainly contained complex proteins with molecular weights of about 55KDa and 60KDa. The complex protein (as shown in Figure 6).

Claims (9)

1. a method for extracting bacterial efflux membrane vesicles, is characterized in that, adopts ultrafiltration concentration method to extract bacterial outer membrane vesicles (OMVs) from Escherichia coli DH5α and Klebsiella pneumoniae bacterium liquid, it comprises: Escherichia coli and Klebsiella pneumoniae were prepared into bacterial suspensions, and an appropriate amount of bacterial suspensions were inoculated into test tubes containing medium for overnight cultivation, and then appropriate amount of bacterial suspensions were inoculated into shake flasks containing medium to cultivate with an OD value close to 1.0. Collect the bacterial liquid, centrifuge to remove the bacterial cells, filter the sterile filter head to remove the residual bacterial cells, collect the filtrate, concentrate it with an ultrafiltration tube with a molecular weight cut-off of 100KDa to 1/8 of the original volume, add physiological saline to wash and repeat this step. 2 to 3 times, the bacterial outer membrane vesicles on the upper layer of the ultrafiltration tube were collected, and placed in a -20°C environment for subpackaging and freezing. 2. the method for extracting bacterial efflux membrane vesicles as claimed in claim 1, is characterized in that, described escherichia coli DH5α is derived from American Culture Collection Center, and Klebsiella pneumoniae is derived from China Agricultural Microorganism Culture Collection center. 3. the method for extracting bacterial efflux membrane vesicles as claimed in claim 1, is characterized in that, described OMVs is the bilayer ball star proteolipid that the form of budding continuously releases on the surface of Escherichia coli and Klebsiella pneumoniae body, carrying the bacterial outer membrane and periplasmic components. 4. the method for extracting bacterial efflux membrane vesicles as claimed in claim 1, is characterized in that, described bacterium liquid refers to making Escherichia coli and Klebsiella pneumoniae bacterium suspension, first takes 10 μ L bacterium suspension coli in LB medium, Klebsiella pneumoniae in NB medium, cultured for 12 hours overnight, respectively transfer the test tube bacterial solution to the corresponding 40ml medium and cultivate until OD value reaches 1.0. 5. the method for extracting bacterial efflux membrane vesicles as claimed in claim 4, is characterized in that, described bacterium liquid is placed in the 50ml centrifuge tube, 6000r/min centrifuges 20min, discards bacterium precipitation and obtains on the culture medium The supernatant was filtered with a 0.22 μM sterile filter head to obtain a sterile filtrate. 6. the method for extracting bacterial efflux membrane vesicles as claimed in claim 1, is characterized in that, described ultrafiltration concentration method comprises, the sterile filtrate that will obtain is added in the ultrafiltration tube of 50ml, 3000r/min centrifugal , the OMVs were retained on the upper part of the ultrafiltration tube, and centrifuged repeatedly until the total volume was concentrated to 1/8 of the original volume, and the OMVs were obtained after washing. 7. the method for extracting bacterial efflux membrane vesicles as claimed in claim 6, is characterized in that, in described cleaning method, adds 5ml normal saline 0.9%NaCl solution in the OMVs that sucrose density gradient centrifugation obtains, changes into Concentrate by ultrafiltration in an ultrafiltration tube and repeat 2-3 times to obtain pure OMV. 8. The use of the bacterial outer membrane vesicles obtained by the method for extracting bacterial efflux membrane vesicles as claimed in claim 1 in the preparation of pharmaceutically acceptable nano drug delivery carriers. 9. The use of the bacterial outer membrane vesicles obtained by the method for extracting bacterial efflux membrane vesicles as claimed in claim 1 in preparing vaccines.