CN111471616A

CN111471616A - Acinetobacter baumannii outer membrane vesicle and preparation method and application thereof

Info

Publication number: CN111471616A
Application number: CN202010265241.4A
Authority: CN
Inventors: 李海波; 李笋; 邹全明; 季露; 陈大群; 曾浩; 张卫军; 鲁东水; 罗萍
Original assignee: Army Medical University
Current assignee: Army Medical University
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2020-07-31

Abstract

The invention relates to the field of Outer Membrane Vesicles (OMVs), and discloses acinetobacter baumannii outer membrane vesicles, a preparation method thereof and application thereof. The method comprises the following steps: carrying out first solid-liquid separation on the fermentation liquor of the acinetobacter baumannii to obtain thallus sediment, and then adding a buffer solution without a detergent into the thallus sediment to destroy the cell walls of the thallus. The method for extracting the outer membrane vesicles from the acinetobacter baumannii does not use any detergent, has high yield of extracting the outer membrane vesicles, and has better effect on protecting mice from being infected by the acinetobacter baumannii.

Description

Acinetobacter baumannii outer membrane vesicle and preparation method and application thereof

Technical Field

The invention relates to the field of outer membrane vesicle OMV, in particular to a method for extracting outer membrane vesicles from acinetobacter baumannii, the acinetobacter baumannii outer membrane vesicles prepared by the method, and the method for extracting the outer membrane vesicles from the acinetobacter baumannii or application of the acinetobacter baumannii outer membrane vesicles in preparation of medicines for preventing and/or treating acinetobacter baumannii infection.

Background

Acinetobacter baumannii (a. baumannii) is a gram-negative bacterium which is generally harmless to healthy individuals, but can cause severe infections in severe hospital patients, such as ventilator-associated pneumonia, wound infection, bacteremia, and the like, and has become one of the main pathogens of nosocomial infections. The acinetobacter baumannii is easy to resist toxic agents and antibiotics, so that great challenge is brought to clinical anti-infection treatment, and the life safety of patients is seriously threatened.

Vaccines are the most economical and effective means of controlling pathogen epidemic infection. A variety of forms of acinetobacter baumannii vaccines are currently being studied, including Inactivated Whole Cell (IWC), Outer Membrane Complex (OMC), Outer Membrane Vesicles (OMV), and the like. The vaccines in the forms have a certain protection effect on the acinetobacter baumannii infected animals, but are still in the experimental research stage at present, and no acinetobacter baumannii vaccine product is on the market internationally.

Bacterial Outer Membrane Vesicles (OMVs) are spherical in structure, about 50-300nm in diameter, generally contain many of the outer membrane protein components of the original bacterium, are non-reproducible and replicable, and stimulate the body to produce an immune response, and are therefore considered to be candidates for an ideal vaccine, and currently, neisseria meningitidis OMVs have been successfully marketed as vaccine components. Since bacteria spontaneously secrete OMVs into the culture system during growth, the bacteria culture supernatant is usually concentrated by ultrafiltration and then ultracentrifuged to enrich and extract OMVs. This process, known as supernatant extraction, has the major disadvantage of low OMV extraction yield. Another commonly used method is detergent extraction, which is performed by collecting the cells, then using a detergent (e.g., sodium deoxycholate) and an ion chelating agent (e.g., EDTA) to promote the formation of OMVs from the cells, and finally collecting the OMVs by ultracentrifugation. Compared with the supernatant extraction method, the yield of the OMV extracted by the detergent is greatly improved, and the detergent extraction method is used for extracting the OMV in the marketed Neisseria meningitidis vaccine group B. However, detergent extraction still suffers from two problems, one being that the extracted OMVs are relatively prone to aggregation, and the other being that detergent residues are usually present.

The existing research shows that OMV vaccines obtained by different extraction methods have differences in the aspects of form, protein composition and L PS content, and the protection effects are also obviously different.

At present, a majority of Acinetobacter baumannii OMVs reported in the literature are obtained by a supernatant extraction method, and although the OMVs extracted by the method have a remarkable protection effect on Acinetobacter baumannii infection, the yield is low.

Disclosure of Invention

The invention aims to overcome the defect that the yield of OMV extracted from Acinetobacter baumannii in the prior art is low, and provides a method for extracting outer membrane vesicles of bacteria from Acinetobacter baumannii, the outer membrane vesicles of Acinetobacter baumannii prepared by the method, and application of the outer membrane vesicles of Acinetobacter baumannii. The method for extracting the outer membrane vesicles from the acinetobacter baumannii does not use any detergent, has high yield of OMV extraction, and has better effect on protecting mice from acinetobacter baumannii infection.

Based on the above research results, the present invention provides, in one aspect, a method for extracting outer membrane vesicles from acinetobacter baumannii, the method comprising: carrying out first solid-liquid separation on the fermentation liquor of the acinetobacter baumannii to obtain thallus sediment, and then adding a buffer solution without a detergent into the thallus sediment to destroy the cell walls of the thallus.

Preferably, the cell wall of the bacterial cells is disrupted by shearing, and the detergent-free buffer contains Tris-HCl, NaCl and EDTA.

Preferably, the method for disrupting cell walls of the bacterial cells comprises an enzymatic method and an ultrasonic method, and the detergent-free buffer comprises sucrose and EDTA.

In a second aspect, the present invention provides acinetobacter baumannii outer membrane vesicles prepared by the method described above.

In a third aspect, the invention provides a method as described above or use of acinetobacter baumannii outer membrane vesicles as described above in the preparation of a medicament for preventing and/or treating acinetobacter baumannii infection.

The method for extracting the outer membrane vesicles from the acinetobacter baumannii does not use any detergent, the yield of the OMV is high, the protein types in the obtained OMV product can reach more than 300, the outer membrane protein accounts for less than 30% of the total protein amount, and the method has a good effect of protecting mice from the acinetobacter baumannii infection.

In the OMV products (nOMVs) prepared preferably by shearing, the protein types are more than 300, the outer membrane protein accounts for 10-20% of the total protein amount, and the outer membrane protein contains L PS;

in OMV products (SuOMV) prepared preferably by ultrasonication and enzymolysis, the protein species are more than 400, and the envelope protein accounts for 25-30% of the total protein amount, and further contains thallus DNA component.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 transmission electron microscopy observation of sOMV;

FIG. 2 Transmission Electron microscopy observations of SuOMV;

FIG. 3 transmission electron microscopy observation of nOMV;

FIG. 4 SDS-PAGE analysis of three OMVs;

FIG. 5 expression of CD40 following stimulation of BMDCs with three different OMVs;

FIG. 6 expression of CD80 following stimulation of BMDCs with three different OMVs;

FIG. 7 expression of CD86 following stimulation of BMDCs with three different OMVs;

FIG. 8 expression of TNF- α in the supernatants of BMDCs cultures following stimulation with three different OMVs;

FIG. 9 expression of I L-1 β in BMDCs culture supernatant following stimulation with three different OMVs;

FIG. 10 expression of I L-6 in the supernatants of BMDCs cultures following stimulation with three different OMVs;

FIG. 11 expression of I L-12 p70 in BMDCs culture supernatant following stimulation with three different OMVs;

FIG. 12 expression of I L-10 in the supernatants of BMDCs cultures following stimulation with three different OMVs;

FIG. 13 protective effects of intramuscular immunization of three different OMVs on the Acinetobacter baumannii pneumonia infection model;

FIG. 14 protective effect of three different OMVs on Acinetobacter baumannii pneumonia infection model by nasal drop immunization;

FIG. 15 specific IgG levels in serum following intramuscular immunization of three different OMVs;

FIG. 16 specific IgG levels in serum following nasal drip immunization of three different OMVs;

FIG. 17 levels of specific sIgA in vaginal lavage following intramuscular immunization of three different OMVs;

FIG. 18 levels of specific sIgA in saliva following intramuscular immunization of three different OMVs;

FIG. 19 levels of specific sIgA in vaginal lavage following nasal drop immunization of three different OMVs;

FIG. 20 levels of specific sIgA in saliva following nasal drip immunization of three different OMVs;

FIG. 21 levels of specific IgG1 in serum following intramuscular immunization of three different OMVs;

FIG. 22 levels of specific IgG2a in serum following intramuscular immunization of three different OMVs;

FIG. 23 levels of specific IgG1 in serum following nasal drip immunization of three different OMVs;

FIG. 24 levels of specific IgG2a in serum following nasal drip immunization of three different OMVs;

FIG. 25 ratio of IgG2a to IgG1 in serum following intramuscular immunization of three different OMVs;

FIG. 26 ratio of IgG2a to IgG1 in serum after nasal drip immunization of three different OMVs.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the present invention provides a method for extracting outer membrane vesicles from acinetobacter baumannii, the method comprising: carrying out first solid-liquid separation on the fermentation liquor of the acinetobacter baumannii to obtain thallus sediment, and then adding a buffer solution without a detergent into the thallus sediment to destroy the cell walls of the thallus.

The invention provides a method for extracting OMV of Acinetobacter baumannii with specificity, which directly extracts OMV from thalli without adopting a detergent, overcomes the defects of detergent residue and easy OMV aggregation caused by the traditional method for extracting OMV by adopting the detergent, and greatly improves the yield of OMV. And the OMV prepared by the invention can more effectively protect mice from being infected by Acinetobacter baumannii.

As is well known, the term "soil release agent" is a substance which can clean soil under ordinary washing conditions, and the soil release agents on the market often contain a surfactant. The process of the present invention may be carried out in the presence or absence of a surfactant, and more preferably, it is not carried out in the presence of a surfactant.

According to another preferred embodiment of the invention, the detergent is any detergent, including ionic as well as non-ionic.

According to the invention, the detergent is preferably a deoxycholate, e.g. sodium deoxycholate.

According to a preferred embodiment of the invention, the method of the invention does not require the use of detergents not only during the cell wall disruption process, but also during the entire extraction of outer membrane vesicles from acinetobacter baumannii.

According to the present invention, the method for breaking the cell wall of the bacteria may employ a conventional cell-breaking method in the art, for example, but not limited to, at least one of enzymolysis, ultrasound, shearing, grinding, crushing, microfluidization, air-fortification and osmotic shock, and it is understood that one of the cell-breaking methods may be used alone, or a combination of 2 or more cell-breaking methods may be used.

According to a preferred embodiment of the present invention, the cell walls of the cells are disrupted by shearing. In the case of the shearing method, the buffer solution containing no detergent may be any of the conventional buffers as long as it does not contain a detergent. Preferably, however, to further increase the OMV yield, the detergent-free buffer comprises, more preferably consists of, Tris-HCl, NaCl and EDTA. Wherein the concentration of Tris-HCl, NaCl and EDTA in the buffer solution can be selected in a wide range, but preferably, in order to further improve the yield of OMV, the concentration of Tris-HCl is 0.01-0.2M, preferably 0.01-0.05M; the concentration of NaCl is 0.05-0.3M, preferably 0.1-0.2M; the concentration of EDTA is 0.005-0.05M, preferably 0.005-0.015M; the pH value of the buffer solution is 6-9, preferably 7-7.5.

According to the present invention, the amount of the buffer solution to be used may be selected from a wide range as long as it can sufficiently shear the bacterial cells, and is preferably 1 to 10ml, more preferably 2 to 3ml, per 1g of the bacterial cell precipitate.

According to the present invention, the mode of shearing the cells is not particularly limited, but it is preferable that the cells are sheared and dispersed by a high-speed dispersion method, specifically, the buffer solution is added to the cell pellet to obtain a cell suspension, and then the cell suspension is sheared and dispersed in a high-speed disperser.

Wherein, the conditions of the shear dispersion can be selected in a wide range, which further improves the yield of OMV, and preferably, the conditions of the shear dispersion comprise: the temperature is 20-40 ℃, preferably 25-35 ℃, the shear rate is 8000-25000g, preferably 15000-20000g, and the time is 1-10min, preferably 2-4 min.

Preferably, in order to further improve the yield of OMVs, the method further comprises incubating the bacterial suspension before shearing the bacterial suspension, wherein the incubation conditions are preferably as follows: the temperature is 50-60 deg.C, preferably 53-58 deg.C, and the time is 20-40min, preferably 25-35 min.

According to the invention, after the cell wall of the thallus is broken, the method further comprises the step of carrying out second solid-liquid separation on the wall-broken product to obtain a crude product of the acinetobacter baumannii outer membrane vesicle.

Although the purpose of the present invention can be achieved by only subjecting the wall-broken product to solid-liquid separation to obtain a solid phase containing acinetobacter baumannii outer membrane vesicles, the inventors of the present invention found in the course of research that the solid-liquid separation is performed by centrifugation, and the centrifugation includes a first centrifugation, a second centrifugation, a third centrifugation and a fourth centrifugation performed in sequence, which can further improve the effect of the prepared OMV product in protecting mice from acinetobacter baumannii infection. Wherein the rotating speed of the centrifugation is sequentially increased along with the centrifugation times.

Preferably, the first centrifugation comprises: centrifuging the wall-broken product at 10000-.

Preferably, the second centrifugation comprises: centrifuging the first supernatant at 20000-30000g (preferably 23000-28000g) at 2-6 deg.C for 10-20min to obtain a second supernatant.

Preferably, the third centrifugation comprises: and centrifuging the second supernatant at 30000-50000g (preferably 30000-35000g) and at 2-6 deg.C for 15-25min to obtain a third supernatant.

Preferably, the fourth centrifugation comprises: and centrifuging the third supernatant at 50000-120000g (80000-120000g) at 2-6 ℃ (1.5-2.5h), and collecting precipitates to obtain a crude product of the acinetobacter baumannii outer membrane vesicles.

According to the present invention, in order to further improve the OMV yield, it is preferable that in the first centrifugation step, after the completion of centrifugation, a supernatant and a pellet are obtained, then the pellet is centrifuged again under the conditions of the first centrifugation treatment, and the supernatants obtained by the two centrifugations are combined to obtain a first supernatant.

According to the present invention, in order to further improve the purity of the OMV product, the method of the present invention preferably further comprises washing the crude acinetobacter baumannii outer membrane vesicles to obtain a pure acinetobacter baumannii outer membrane vesicles. The washing method may be performed according to a method conventional in the art, for example, the crude acinetobacter baumannii outer membrane vesicle is washed in an insoluble washing solution, the washing solution may be one of water, a PBS buffer solution and a sucrose solution, and the number of washing may be 1 or more. According to a preferred embodiment of the present invention, the crude Acinetobacter baumannii outer membrane vesicles are resuspended in PBS buffer, and centrifuged (1.5-2.5h) at 20000-.

According to a preferred embodiment of the invention, after Acinetobacter baumannii is cultured to the late logarithmic growth stage, the bacterial liquid is centrifuged, and the thallus is collected; the thallus is dispersed in a buffer solution containing 0.01-0.05M Tris-HCl, 0.1-0.2M NaCl, 0.005-0.015M EDTA and pH 7-7.5, and the ratio of wet weight of the wet thallus to the volume of the buffer solution is 1:2-3 (g/ml); incubating at 53-58 deg.C for 25-35min, and cooling to room temperature. Dispersing at room temperature with high speed disperser 15000-20000g for 2-4min, centrifuging at 2-6 deg.C with 12000-18000g for 10-20min, dispersing the precipitate with high speed disperser, and repeating the above centrifuging steps. Combining the supernatants obtained by the two centrifugations, centrifugating for 10-20min at 2-6 ℃ by 23000g, centrifugating for 15-25min at 2-6 ℃ by 30000g and 35000g, centrifugating the supernate again for 1.5-2.5h at 2-6 ℃ by 80000g and 120000g, and collecting the precipitate to obtain crude OMV; the crude product is dispersed in PBS for washing, 20000-ion 50000g is centrifuged for 1.5-2.5h at the temperature of 2-6 ℃, and the precipitate is the pure product of Acinetobacter baumannii OMV.

The OMV thus obtained is abbreviated as nOMV, has a nearly spherical shape and uniform size, has an average particle diameter of 217.45-286.73nm (PDI:0.086), contains more than 300 kinds of proteins, has an outer membrane protein content of 70-80% of the total protein content, and contains no DNA.

According to another preferred embodiment of the present invention, the cell walls of the cells are disrupted by a combination of enzymatic and ultrasonic methods, and in this preferred case, the buffer solution containing no detergent may be any of various conventional buffer solutions as long as it contains no detergent. Preferably, however, to further increase the OMV yield, the detergent-free buffer comprises, more preferably consists of, sucrose, Tris-HCl, EDTA and lysozyme. Wherein the concentration of sucrose, Tris-HCl, EDTA and lysozyme in the buffer may be selected within a wide range, but preferably, in order to further increase the yield of OMVs, the concentration of sucrose is 0.05-1M, preferably 0.5-0.75M; the concentration of Tris-HCl is 0.01-0.2M, preferably 0.01-0.05M; the concentration of EDTA is 1-5m M, preferably 1-2 mM; the concentration of the lysozyme is 0.05-20mg/ml, preferably 5-15 mg/ml; the pH value of the buffer is 6-9, preferably 7-8.

According to the present invention, the amount of the buffer solution to be used may be selected from a wide range as long as the bacterial cells can be sufficiently suspended, and it is preferable that the amount of the buffer solution to be used is 15 to 100ml per 1g of the bacterial cell pellet.

According to the present invention, although the subsequent ultrasonication can be performed after resuspending the pellet of the cells using a buffer containing sucrose, Tris-HCl, EDTA and lysozyme, it is preferable that the method of disrupting the cell walls of the cells using the enzymatic method and the ultrasonic method comprises: adding a first sucrose solution into the thallus precipitate to obtain a heavy-suspension bacterium solution, then adding lysozyme and EDTA into the heavy-suspension bacterium solution, and carrying out ultrasonic wall breaking on the obtained mixed material. Wherein the first sucrose solution contains sucrose and Tris-HCl, preferably consists of sucrose and Tris-HCl.

According to the present invention, the amount of the first sucrose solution to be used may be selected from a wide range as long as the microbial cells can be sufficiently suspended, but in order to further improve the yield of OMVs, it is preferable that the amount of the first sucrose solution to be added is 15 to 100ml, preferably 15 to 25ml, and more preferably 18 to 22ml, relative to 1g of the microbial cell precipitate; preferably, the first sucrose solution contains 0.05-1M, preferably 0.5-0.75M sucrose, and 0.01-0.2M, preferably 0.01-0.05M Tris-HCl.

According to the present invention, the amount of lysozyme added may be selected from a wide range, as long as it is effective in combination with ultrasonic treatment to sufficiently break the cell walls of the cells, but in order to further improve the yield of OMV, it is preferable that the amount of lysozyme added is such that the concentration of lysozyme in the mixed material is 0.05-20mg/ml, preferably 5-15 mg/ml. Here, the cell pellet and lysozyme volume were ignored.

According to the present invention, the amount of EDTA to be added may be selected from a wide range as long as it is effective in combination with sonication to sufficiently break the cell walls of the cells, but in order to further improve the yield of OMV, it is preferable that the amount of EDTA to be added is such that the concentration of EDTA in the mixture is 1 to 5mM, preferably 1 to 2 mM. Here, the cell pellet and lysozyme volume were ignored.

According to the present invention, the conditions for the ultrasonic wall breaking may also be selected within a wide range, but in order to further improve the yield of OMVs, it is preferred that the ultrasonic wall breaking is performed in an ice bath for 5-15min, wherein the break is stopped for 4-6s per 3-5s of ultrasonication.

According to the invention, after the cell wall of the thallus is broken, the method further comprises the step of carrying out third solid-liquid separation on the wall-broken product to obtain a crude product of the acinetobacter baumannii outer membrane vesicle.

Although the purpose of the present invention can be achieved by only subjecting the wall-broken product to solid-liquid separation to obtain a solid phase containing acinetobacter baumannii outer membrane vesicles, the inventors of the present invention found in the course of research that the solid-liquid separation is performed by centrifugation, and the centrifugation includes a first centrifugation and a second centrifugation performed in sequence, which can further improve the effect of the prepared OMV product in protecting mice from acinetobacter baumannii infection. Preferably, the first centrifugation is used for removing large-particle impurities and unbroken thalli in the wall-broken product, and the second centrifugation is used for collecting precipitates to obtain a crude product of the acinetobacter baumannii outer membrane vesicles.

Preferably, the first centrifugation comprises: the wall-breaking product is centrifuged (10-20min) at the temperature of 1000-2000g (preferably 1000-1500g) and 2-6 ℃ to obtain a first supernatant.

Preferably, the second centrifugation comprises: and centrifuging the first supernatant at 200000-500000g (preferably 300000-400000g) at 2-6 ℃ (1.5-2.5h), and collecting precipitate to obtain crude product of acinetobacter baumannii outer membrane vesicles.

In order to further increase the purity of the OMV product according to the invention, the process of the invention preferably further comprises: and (3) resuspending the crude product of the acinetobacter baumannii outer membrane vesicle in a second sucrose solution, then carrying out sucrose density gradient centrifugation, and collecting a sucrose layer containing the acinetobacter baumannii outer membrane vesicle. Wherein the second sucrose solution contains sucrose and EDTA. The concentrations of sucrose and EDTA can be selected within a wide range, as long as the crude acinetobacter baumannii outer membrane vesicles can be effectively resuspended and subjected to subsequent sucrose density gradient centrifugation. Preferably, the second sucrose solution contains 20-30 wt.%, preferably 22-28 wt.% sucrose and 4-6mM, preferably 4.5-5.5mM EDTA, pH 7-8.

Wherein, preferably, the sucrose density gradient of the sucrose density gradient centrifugation is 30-55 wt%, and the sucrose layer containing the acinetobacter baumannii outer membrane vesicle is 45-50 wt% gradient sucrose layer. Wherein the sucrose density gradient may be set to 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt% and 55 wt% in this order, wherein the sucrose solution of each sucrose layer contains sucrose and EDTA in respective amounts, wherein the EDTA content in each sucrose layer is 4-6mM, preferably 4.5-5.5mM, independently of each other, and the pH is 7-8, independently of each other.

Wherein, the conditions of the sucrose density gradient centrifugation can be selected in a wide range, in order to improve the yield of OMV, the centrifugation treatment (10-20h) is preferably carried out under the conditions of 200000-500000g (preferably 300000-400000g) and 2-6 ℃, and after the centrifugation is finished, a 45-50 weight percent sucrose layer containing the acinetobacter baumannii outer membrane vesicles is collected.

According to the present invention, preferably, the method further comprises diluting the sucrose layer containing acinetobacter baumannii outer membrane vesicles, and then centrifuging and collecting the precipitate to obtain a pure product of acinetobacter baumannii outer membrane vesicles. Wherein the sucrose layer containing acinetobacter baumannii outer membrane vesicles may be diluted with a conventional buffer, but preferably, the sucrose layer containing acinetobacter baumannii outer membrane vesicles is diluted with EDTA in an amount of 0.5 to 1ml in terms of 1mM of EDTA (pH7 to 8) with respect to 1ml of the sucrose layer in order to further improve OMV yield. The rotation speed of the centrifugation is preferably 40000-80000g, and the time is preferably 1.5-2.5 h.

According to the present invention, in order to further enhance the immune effect of the prepared OMV, preferably, before the crude acinetobacter baumannii outer membrane vesicles are resuspended in the second sucrose solution, the method further comprises: and washing the crude product of the acinetobacter baumannii outer membrane vesicle in a third sucrose solution, and then re-suspending the washed solid phase in a second sucrose solution, wherein the third sucrose solution contains sucrose, Tris-HCl and EDTA.

Preferably, the third sucrose solution contains 0.2-0.3M sucrose, 3-4mM Tris-HCl, 0.8-1.2mM EDTA, and has a pH of 7.5-8.

Preferably, the washed solid phase is obtained by centrifugation at 200000-.

According to a preferred embodiment of the invention, Acinetobacter baumannii is cultured to the late logarithmic growth stage, the bacterial liquid is centrifuged, and the thallus is collected; the cells were immediately resuspended in a pre-chilled first sucrose solution (0.5-0.75M sucrose, 0.01-0.05M Tris-HCl, pH7.5-8) at a wet cell weight to buffer volume ratio of 1:18-22 (g/ml). Lysozyme (lysozyme) was added to the resuspended suspension at 5-15mg per ml. Next, a pre-cooled 1.5mM EDTA solution was further added thereto so as to have a concentration of 1 to 2mM, and the mixture was gently stirred and mixed uniformly. And (4) carrying out ultrasonic treatment by an ultrasonic crusher for 5-15min in ice bath (3-5 seconds and 4-6 seconds for stopping every time).

Centrifuging the suspension for 10-20min at 2-6 ℃ and 1500- × g after ultrasonic treatment for 1000- × g, removing uncrushed thallus, re-suspending the supernatant by 300000-400000g and 40056 g at 2-6 ℃ for 1.5-2.5h, collecting the precipitate, centrifuging the precipitate for 1.5-2.5h by 300000-400000g and 2-6 ℃ for 1.5-2.5h by using a third sucrose solution (0.2-0.3M sucrose, 3-4mM Tris-HCl, 0.8-1.2mM EDTA and pH7-8) and re-suspending the precipitate by using a second sucrose solution (22-28 wt% sucrose, 4.5-5.5mM EDTA and pH7-8), performing sucrose density gradient centrifugation (55%, 50%, 45%, 40%, 35%, 30% sucrose, 4.5-5.5mM EDTA and pH7-8) and then centrifuging the precipitate by using 10-20h as a sucrose density gradient (55%, 45%, 35%, 30% EDTA, 4.5-5 mM EDTA and 5.5% EDTA and 5-5H) by adding 0.00-5 ml of pure sucrose solution (1-5 ml) to the supernatant of Acinetobacterium (10-20h) and then adding 0.00-5 ml of pure sucrose solution (5-5 ml) and diluting the supernatant by using 10-5 mM EDTA and 1.5mM EDTA).

The OMV is called SuOMV for short, and has nearly spherical shape, uniform size, average particle diameter of 106.37-178.69nm, containing more than 400 proteins, and DNA with outer membrane protein content of 50-60 wt% of total protein.

According to the present invention, said acinetobacter baumannii is any acinetobacter baumannii capable of expressing OMVs, preferably said acinetobacter baumannii is acinetobacter baumannii ATCC17978 strain.

In a second aspect, the present invention provides acinetobacter baumannii outer membrane vesicles prepared by the method as described above.

In a third aspect, the invention provides the method or the application of the acinetobacter baumannii outer membrane vesicle in preparation of a medicament for preventing and/or treating acinetobacter baumannii infection.

Examples

The present invention will be described in detail below by way of examples. In the following examples and comparative examples:

acinetobacter baumannii 17978 strain, ATCC17978, cryopreserved by the applicant;

GI1640 medium (cat # SH30809.01) Hyclone;

PBS buffer powder (pH 7.3) (cat # Z L I-9062);

the goat anti-mouse IgG antibody (cat # ZB-2305) of the goat is marked by HRP (Japanese horse radish seed) of China fir bridgework;

l method protein concentration determination kit (1000T) (cat # PC0030) Solaibao;

yeast extract (cat # L P0021), OXOID;

peptone (cat # L P0042), OXOID;

sodium chloride (cat # A610476-9001) BBI;

soluble single-component TMB substrate solution (cat # PA107) of Tiangen organisms;

goat anti-mouse IgG2a heavy chain (HRP) (cat # ab 97245); goat anti-mouse IgG1Heavychain (HRP) (cat # ab97240) abcam;

mouse I L-1 β Elisa Kit (cat No. 1210122), Mouse TNF- α Elisa Kit (cat No. 1217202), Mouse I L-6 Elisakut (cat No. 1210602), Mouse I L-12 p70 Elisa Kit (cat No. 1211202), Mouse I L-10 Elisa Kit (cat No. 1211002) Davidae as organisms;

PCR Forward Primer; PCR Reverse Primer; TaqMan Probe was synthesized in Yingjun organisms;

PE anti-mouse CD40 (cat # 124609); APC anti-mouse CD86 (cat # 105011); PerCP/Cy5.5 anti-mouse CD80 (cat number: 104721); FITC anti-mouse CD11c (cat # 117305) Biolegend.

Examples 1 to 1

This example illustrates the extraction of Acinetobacter baumannii from OMV provided by the invention

Acinetobacter baumannii 17978 strain is liquid cultured in L B (L uri-Bertani) culture medium to the late logarithmic growth stage, the bacterial liquid 6000 × g is centrifuged at 4 ℃ for 40min, the bacterial bodies are collected, the bacterial bodies are dispersed in buffer solution containing 0.05M Tris-HCl, 0.15M NaCl and 0.01M EDTA and having pH of 7.4, the ratio of wet weight of the wet bacteria to the volume of the buffer solution is 1:2.5(M/v), the mixture is incubated at 56 ℃ for 30min and then cooled to room temperature, the mixture is dispersed at 18000 × g for 3min by using a high-speed disperser at room temperature, then 150 × g is centrifuged at 4 ℃ for 15min, the precipitate is dispersed again by using the high-speed disperser under the same conditions, the centrifugation steps are repeated, the supernatants obtained by the two times of centrifugation are combined, 25000g is centrifuged at 4 ℃ for 15min, the supernatant is centrifuged at 30000 × g at 4 ℃ for 20min, 1000 × g of the supernatant obtained by the centrifugation at 4 ℃ for 2h, the precipitate is collected, the crude product obtained by the centrifugation of Acinetobacter baumannii 178, the crude product obtained by the centrifugation of 290.v, the centrifugation of the supernatant obtained by the centrifugation of 100000 nV, and the centrifugation of the final product obtained by the centrifugation.

Examples 1 to 2

Acinetobacter baumannii 17978 strain is subjected to liquid culture in L B (L aria-Bertani) culture medium until the late logarithmic growth stage, the strain is centrifuged for 40min at 4 ℃ by 6000 × g of bacteria liquid, the bacteria liquid is collected, the bacteria liquid is dispersed in buffer solution containing 0.04M Tris-HCl, 0.2M NaCl and 0.005M EDTA and having the pH value of 7.4, the ratio of the wet weight of the wet bacteria to the volume of the buffer solution is 1:2(M/v), the temperature is incubated for 35min at 58 ℃, the temperature is cooled to room temperature, the bacteria liquid is dispersed for 4min by a disperser at 15000g at the room temperature, 12000g is centrifuged for 20min at 4 ℃, the precipitate is dispersed again by a high-speed disperser under the same condition, the centrifugation steps are repeated, the supernatant obtained by combining the two times of centrifugation is centrifuged, 28000g is centrifuged for 10min at 4 ℃, the supernatant is centrifuged for 15min at 32000g at 4 ℃, 80000g of the centrifuged supernatant is centrifuged again at 4 ℃ for 2.5h, the crude product is obtained by collecting the crude product of Acinetobacter baumannii, the OMV is dispersed in PBS, and the OMV is collected by the final centrifugation of 80000g, the OMV is calculated as OMV product, and the OMV product is.

Examples 1 to 3

Acinetobacter baumannii 17978 strain is cultured in L B (L aria-Bertani) culture medium liquid to the late logarithmic growth stage, liquid 6000 × g is centrifuged at 4 ℃ for 40min to collect thalli, the thalli are dispersed in buffer solution containing 0.06M Tris-HCl, 0.1M NaCl and 0.015M EDTA and having pH 7.4, the ratio of wet weight of wet bacteria to volume of the buffer solution is 1:3(M/v), the temperature is incubated at 53 ℃ for 25min, then the temperature is cooled to room temperature, the thalli are dispersed for 2min by using a disperser 20000g at high speed at room temperature, then 18000g is centrifuged at 4 ℃ for 10min, the precipitate is dispersed again by using a high-speed disperser under the same condition, the centrifugation steps are repeated, the supernatant obtained by combining the centrifugation steps, 23000g is centrifuged at 4 ℃ for 20min, the supernatant is centrifuged at 35000g at 4 ℃ for 25min again, the centrifugal supernatant is centrifuged again 120000g at 4 ℃ for 1.5h to obtain precipitate, crude product of nV, the crude product is obtained by dispersing in OMV, and the OMV is collected by washing, the OMV is finally collected by OMV, and the OMV, the OMV is calculated as OMV product, wherein the OMV is calculated by using the OMV for 512-512 mg.

Examples 1 to 4

Acinetobacter baumannii 17978 strain is cultured by L B (L ura-Bertani) culture medium liquid to the late logarithmic growth stage, the bacterium liquid 6000g is centrifuged for 40min at 4 ℃, the bacterium is collected, the bacterium is dispersed in buffer solution containing 0.1M Tris-HCl, 0.3M NaCl and 0.002MEDTA and having pH of 7.4, the ratio of wet weight of wet bacterium to volume of the buffer solution is 1:10(M/v), the mixture is cooled to room temperature after being incubated for 30min at 60 ℃, the mixture is dispersed for 3min at 25000g by using a high-speed disperser at room temperature, then 10000g is centrifuged for 20min at 4 ℃, the precipitate is dispersed again by using the high-speed disperser under the same condition, the centrifugation steps are repeated, the supernatant obtained by the centrifugation steps are combined, 30000g is centrifuged for 20min at 4 ℃, the supernatant is centrifuged for 20min at 30000g at 4 ℃, 120000g is centrifuged again at 4 ℃ for 2h, the precipitate is collected, the crude product is obtained by dispersing in PBS, washing, the crude product is obtained by centrifuging Acinetobacter baumannii, namely, the final product is collected by centrifugation at 4 ℃ and the centrifugation step of 357, namely, the absolute value of absolute n.

Examples 1 to 5

Acinetobacter baumannii OMV was extracted according to the method of example 1-1, except that the incubation step was not included, and in the centrifugation step, the combined supernatant was centrifuged at 100000g at 4 ℃ for 2h, the precipitate was collected, the precipitate was dissolved again in PBS of the same volume, and then centrifuged at 25000g at 4 ℃ for 15min, the supernatant was centrifuged at 30000g at 4 ℃ for 20min and 29500g at 4 ℃ for 2h, the precipitate was Acinetobacter baumannii OMV-5, and the final product of nOMV was collected at 0.223 mg/g wet bacteria.

Example 2-1

Acinetobacter baumannii 17978 strain is liquid-cultured by L B culture medium to late logarithmic growth, 6000g of bacterial liquid is centrifuged for 1h at 4 ℃, the bacteria are immediately re-suspended by precooler A (0.75M sucrose, 0.01M Tris-HCl, pH 7.8), the ratio of the wet weight of the bacteria to the volume of Buffer solution is 1:20(M/v), 10mg/M L concentration of lysozyme (lysozyme) is dropwise added into the re-suspended bacterial liquid, 10mg per milliliter is added, then, precooled EDTA solution is continuously added into the re-suspended bacterial liquid to enable the concentration to be 1.5mM, the mixture is slowly stirred for 10min, and ultrasonic crushing is carried out by an ultrasonic crusher for 10min (4 s per work and 5s stop) under ice bath.

Centrifuging the suspension at 4 deg.C for 15min at 1200g after ultrasonic treatment to remove un-broken thallus, centrifuging the supernatant at 4 deg.C for 2h at 360000g, and collecting precipitate. The pellet was resuspended in Buffer B (0.25M sucrose, 3.3mM Tris-HCl, 1mM EDTA, pH 7.8) to the pre-centrifugation volume, and the pellet was collected by centrifugation at 360000g for 2h at 4 ℃. The pellet was resuspended in Buffer C (25 wt% sucrose, 5mM EDTA, pH 7.5) and then subjected to sucrose density gradient centrifugation (55 wt%, 50 wt%, 45 wt%, 40 wt%, 35 wt%, 30 wt% sucrose, 5mM EDTA, pH 7.5), 360000g, centrifugation at 4 ℃ for 16 h. The second layer (45-50 wt% sucrose layer) was collected, diluted with 1mM EDTA, pH7.5 solution, and centrifuged at 60000g for 2h to precipitate Acinetobacter baumannii SuOMV-1. 3.382mg of the SuOMV end product can be collected per gram of wet bacteria.

Examples 2 to 2

Acinetobacter baumannii 17978 strain is liquid-cultured by L B culture medium to late logarithmic growth, 6000g of bacterial liquid is centrifuged for 1h at 4 ℃, the bacteria are immediately re-suspended by precooler A (0.6M sucrose, 0.05M Tris-HCl, pH 7.8), the ratio of the wet weight of the bacteria to the volume of Buffer solution is 1:18(M/v), 10mg/M L concentration of lysozyme (lysozyme) is dropwise added into the re-suspended bacterial liquid, 5mg per milliliter is added, then, precooled EDTA solution is continuously added into the re-suspended bacterial liquid to enable the concentration to be 1mM, the mixture is slowly stirred for 10min, and ultrasonic treatment is carried out by an ultrasonic crusher under ice bath for 8min (5 seconds per working and 4 seconds).

Centrifuging the suspension at 4 deg.C for 10min at 1500g after ultrasonic treatment to remove un-broken thallus, centrifuging the supernatant at 4 deg.C for 1.5h at 400000g, and collecting precipitate. The pellet was resuspended in Buffer B (0.2M sucrose, 3mM Tris-HCl, 1.2mM EDTA, pH 7.8) to the pre-centrifugation volume, and the pellet was collected by centrifugation at 400000g for 1.5h at 4 ℃. The pellet was resuspended in Buffer C (25 wt% sucrose, 5mM EDTA, pH 7.5) and then subjected to sucrose density gradient centrifugation (55 wt%, 50 wt%, 45 wt%, 40 wt%, 35 wt%, 30 wt% sucrose, 4.5mM EDTA, pH 7.5), 400000g, centrifugation at 4 ℃ for 12 h. The second layer (45-50 wt% sucrose layer) was collected, diluted with 1mM EDTA, pH7.5 solution, and centrifuged at 60000g for 2h to precipitate Acinetobacter baumannii SuOMV-2. 2.782mg of the final SuOMV product can be collected per gram of wet bacteria.

Examples 2 to 3

Acinetobacter baumannii 17978 strain is liquid-cultured by L B culture medium to late logarithmic growth, 6000g of bacterial liquid is centrifuged for 1h at 4 ℃, the bacteria are immediately re-suspended by precooler A (0.5M sucrose, 0.03M Tris-HCl, pH 7.8), the ratio of the wet weight of the bacteria to the volume of Buffer solution is 1:22(M/v), 10mg/M L concentration of lysozyme (lysozyme) is dropwise added into the re-suspended bacterial liquid, 15mg per ml is added, then, precooled EDTA solution is continuously added into the re-suspended bacterial liquid to enable the concentration to be 2mM, the mixture is slowly stirred for 10min, and ultrasonic treatment is carried out by an ultrasonic crusher for 12min (3 s per working and 6s per second) under ice bath.

Centrifuging the suspension at 4 deg.C for 20min at 1000g after ultrasonic treatment to remove un-broken thallus, centrifuging the supernatant at 4 deg.C for 2.5h at 300000g, and collecting precipitate. The pellet was resuspended in Buffer B (0.2M sucrose, 4mM Tris-HCl, 0.8mM EDTA, pH 7.8) to the pre-centrifugation volume, centrifuged at 300000g at 4 ℃ for 2.5h, and the pellet was collected. The pellet was resuspended in Buffer C (25 wt% sucrose, 5mM EDTA, pH 7.5) and then subjected to sucrose density gradient centrifugation (55 wt%, 50 wt%, 45 wt%, 40 wt%, 35 wt%, 30 wt% sucrose, 5mM EDTA, pH 7.5), 300000g, at 4 ℃ for 20 h. The second layer (45-50 wt% sucrose layer) was collected, diluted with 1mM EDTA, pH7.5 solution, and centrifuged at 60000g for 2h to precipitate Acinetobacter baumannii SuOMV-3. SuOMV end product 3.124 mg/g wet bacteria can be collected.

Examples 2 to 4

Acinetobacter baumannii 17978 strain is liquid-cultured by L B culture medium to late logarithmic growth, 6000g of bacterial liquid is centrifuged for 1h at 4 ℃, the bacteria are immediately re-suspended by precooler A (0.8M sucrose, 0.07mM Tris-HCl, pH 7.8), the ratio of the wet weight of the bacteria to the volume of Buffer solution is 1:15(M/v), 10mg/M L concentration of lysozyme (lysozyme) is dropwise added into the re-suspended bacterial liquid, 20mg per milliliter is added, then, precooled EDTA solution is continuously added into the re-suspended bacterial liquid to enable the concentration to be 0.5mM, the mixture is slowly stirred for 10min, and ultrasonic crushing is carried out by an ultrasonic crusher for 10min (4 s per work and 5s stop) under ice bath.

Centrifuging the suspension at 4 deg.C for 15min at 2000g after ultrasonic treatment to remove un-broken thallus, centrifuging the supernatant at 4 deg.C for 2h at 500000g, and collecting precipitate. The pellet was resuspended in Buffer B (0.25M sucrose, 3.3mM Tris-HCl, 1mM EDTA, pH 7.8) to the pre-centrifugation volume, and the pellet was collected by centrifugation at 500000g for 2h at 4 ℃. The pellet was resuspended in Buffer C (25 wt% sucrose, 5mM EDTA, pH 7.5) and then subjected to sucrose density gradient centrifugation (55 wt%, 50 wt%, 45 wt%, 40 wt%, 35 wt%, 30 wt% sucrose, 5mM EDTA, pH 7.5), 400000g, at 4 ℃ for 16 h. The second layer (45-50 wt% sucrose layer) was collected, diluted with 1mM EDTA, pH7.5 solution, and centrifuged at 60000g for 2h to precipitate Acinetobacter baumannii SuOMV-4. 1.343mg of SuOMV final product can be collected per gram of wet bacteria.

Examples 2 to 5

Extraction of Acinetobacter baumannii OMV (SuOMV-5) was carried out in the same manner as in example 1-1, except that 0.752mg of SuOMV end product per gram of wet bacteria was collected without adding lysozyme.

Examples 2 to 6

Extraction of Acinetobacter baumannii OMV (SuOMV-6) was performed according to the method of example 1-1, except that ultrasonication was not performed. 0.609mg of SuOMV end product can be collected per gram of wet bacteria.

Examples 2 to 7

The extraction of Acinetobacter baumannii OMV (SuOMV-7) was performed according to the method of example 1-1, except that the step of washing the precipitate with Buffer B was not included. 0.717mg of the final product of SuOMV can be collected per gram of wet bacteria.

Comparative example 1

This comparative example illustrates the extraction of a reference A.baumannii OMV

Acinetobacter baumannii 17978 strain is inoculated and cultured by a three-line method, 10L bacterial liquid is obtained after secondary activation, 6000g of bacterial liquid is centrifuged for 40min, the supernatant is taken, the supernatant is filtered by a 0.22um filter membrane and then concentrated by a 100KD hollow fiber column which is cleaned to be neutral in advance, the concentrated solution is washed by PBS, 200ml of concentrated solution is finally collected, the concentrated solution is centrifuged at a high speed of 150000g and at a temperature of 4 ℃ for 2h, and precipitates are collected, so that Acinetobacter baumannii sOMV is obtained, and 0.128mg of sOMV final product can be collected per gram of wet bacteria.

Test example 1

Acinetobacter baumannii OMV morphology and particle size analysis

Acinetobacter baumannii OMVs obtained in example 1-1(nOMV-1), example 2-1(SuOMV-1) and comparative example 1(sOMV) were prepared into a solution with a mass concentration of 0.1mg/m L, dropped on a carbon film-covered electron microscope copper mesh, excess liquid was sucked off by filter paper, 2 wt% uranyl acetate was dropped for dyeing, and after air-drying, the morphology was observed by a transmission electron microscope, as shown in FIGS. 1-3, all three Acinetobacter baumannii OMVs were approximately spherical and uniform in size.

Measuring the particle sizes of OMVs of three Acinetobacter baumannii by using a dynamic light scattering instrument, wherein the average particle size of sOMV is 183.3nm (PdI: 0.364); SuOMV has an average particle size of 142.9nm (PdI: 0.285); the average particle diameter of nOMV was 269.9nm (PdI: 0.086).

Test example 2

Acinetobacter baumannii OMV protein composition analysis

SDS-PAGE showed that the proteins with the highest content in Acinetobacter baumannii OMVs extracted in example 1-1(nOMV-1), example 2-1(SuOMV-1) and comparative example 1(sOMV) were all OmpA, and the protein compositions of the three were similar (FIG. 4). MA L DI-TOF mass spectrometry showed that sOMV contained 246 proteins, that SuOMV contained 454 proteins, and that nOMV contained 397 proteins.

Test example 3

L PS and DNA content determination in Acinetobacter baumannii OMV

L PS content determination is performed by 3-deoxy-D-manno-2-octulosonacid (3-deoxy-D-mano-oct-2-ulopyranosonicacid, Kdo) method 50 μ L of each of L PS sample and Kdo standard (0,0.05,0.1,0.2,0.3,0.4,0.5mM Kdo) is added into a centrifuge tube, 60 μ L0.018 0.018. 0.018M H is added₂SO₄(0.009M), mixing, standing at 100 deg.C for 20min, heating for hydrolysis, cooling to room temperature, adding 25 μ l of 9.1mg/M L periodic acid, dissolving in 0.125M H₂SO₄I.e. 0.0625M), mixed well and incubated for 20min in the absence of light. Adding 50 μ l of arsenous acid(2.6％NaAsO₂Adding the mixture into 0.5M HCl), stirring until light yellow disappears, adding 250M L0.3.3 percent TBA reagent, mixing by vortex, standing at 100 ℃ for 10 minutes, adding 125 mu L DMSO while the mixture is hot, mixing fully, cooling to room temperature, adding 200 mu L into each hole of an ELISA plate, measuring the absorbance value of A550, and measuring the result to show that the content of L PS in sOMV is 0.139 nmol/mu g, the content of L PS in SuOMV is 0.103 nmol/mu g and the content of L PS in nOMV is 0.417 nmol/mu g according to Kdo^TM

The dsDNA kit measures the DNA content in OMV extracted by three different methods, no DNA is detected in sOMV and nOMV, and the DNA content in SuOMV is 0.4 ng/. mu.g.

Test example 4

Evaluation of activation of bone marrow-derived dendritic cells (BMDCs) by OMV

Preparation of BMDCs:

killing a C57 mouse by cervical dislocation, placing a femur into 1640 culture medium under aseptic condition, sucking the culture medium by using a 1ml sterile syringe, flushing out bone marrow from one end of the femur, grinding and filtering by using a 200-mesh sterilized stainless steel screen, transferring into a 15ml centrifuge tube, centrifuging for 5min at 1800g, discarding supernatant, adding 5ml erythrocyte lysate, standing at room temperature for 2-3min, adding sterile PBS to 10ml, centrifuging for 5min at 1800g, discarding supernatant, washing once by using PBS, re-suspending precipitate by using 1ml complete culture medium (1640: cyan-streptomycin 100:10:1), counting, and re-suspending according to 1 × 10⁶Placing in six-well plate at 37 deg.C and 5% CO, adding GM-CSF and I L-4 to complete culture medium, adjusting to 20ng/ml, placing in six-well plate₂Culturing in incubator to the third day, sucking out the culture medium after gentle blowing, replacing the complete culture medium, and supplementing GM-CSF to 20 ng/ml. Culture to the fifth day, half the fluid change, and make up GM-CSF to 20 ng/ml. Culturing to the seventh day, and collecting suspension cells to obtain the BMDCs.

Flow cytometry detection:

8ug nOMV were added to BMDCs in parallel wells. Standing at 37 deg.C for 5% CO₂Cells were harvested after 24h incubation in an incubator. Gently blow and beat the cells and transfer themThe cells are resuspended by 100ul of staining buffer, and after antibodies 0.2 mu L (anti-CD11c, anti-CD40, anti-CD80 and anti-CD86) are respectively added and stained in dark for 30min, the BMDCs maturation condition is detected by flow type, the result is shown in figure 5-7, and compared with the control group PBS, sOMV, SuOMV and nOMV can obviously increase the expression of CD40, CD80 and CD86 on the surface of the BMDCs, which indicates that three Acinetobacter baumannii OMVs can promote the maturation of the DCs and the effect of SuOMV is optimal (the effect of 2 SuOMVs in the three detection indexes is optimal).

Test example 5

Cytokine determination in BMDCs culture supernatant following OMV stimulation

After BMDCs were stimulated by three OMVs, cell culture supernatants were collected, Mouse I L-1 β E β ISA Kit, Mouse I β 1-6E β ISA Kit, Mouse I β -10E β ISA Kit, Mouse TNF- β E L ISA Kit, and Mouse I L-12 p 70E L ISAKit examined cytokine expression in the supernatant the results are shown in FIGS. 8-12, sOMV, SuOMV, and nOMV both stimulated significant upregulation of I L-1 β and TNF- α compared to the control group, further suggesting that Baobacillus OMV could promote maturation of DCs and activate innate immune response, furthermore, three OMVs could significantly induce upregulation of I L-6 and I L-12 p70, suggesting that Th17 and Th 5 mediated immunoregulation could be activated, and that OMV L-2 mediated responses could be activated.

Test example 6

Grouping and immunization

Experimental animals: c57 mouse, female, 6-8 weeks old (Beijing Huafukang)

Grouping experiments: sOMV intramuscular injection group, SuOMV intramuscular injection group, nOMV intramuscular injection group, sOMV nasal drop group, SuOMV nasal drop group, nOMV nasal drop group, PBS group, 7 groups, each group consisting of 7 mice. The final concentration of OMV was 20. mu.g for each group.

The experimental scheme is as follows:

the mice were immunized by intramuscular injection and nasal drip respectively on days 0, 14 and 28, after the last immunization for 14 days, the challenge was carried out by tracheal intubation with Acinetobacter baumannii L ac-4 strain, 1 day before each immunization and one day before challenge, mouse saliva, vaginal lavage fluid and tail vein blood were collected, centrifuged for 15min at 3000g at 4 ℃ and serum was collected in a sterile EP tube, all samples were stored at-80 ℃ for standby application, and the challenge protection results show that the protection rates of sOMV, SuOMV and nOMV are respectively 50%, 62.5% and 37.5% (FIG. 13) by the intramuscular injection immunization route, the protection rate of SuOMV is 100%, the protection rate of sOMV is 66.7% and the protection rate of nOMV is 50% (FIG. 14) by the nasal drip immunization route.

Test example 7

Detection of specific antibody levels

Solution preparation:

dissolving 1.59g of sodium carbonate and 2.93g of sodium bicarbonate in 1L primary water to prepare a coating buffer solution with the pH value of 9.6, dissolving 0.5ml of Tween-20 in 1L PBS to prepare PBST lotion, uniformly mixing PBST and casein according to the ratio of 100ml to 0.5g to prepare an antibody diluent, uniformly mixing PBS and casein according to the ratio of 100ml to 1g to prepare a confining liquid, and uniformly mixing concentrated sulfuric acid and primary water according to the ratio of 1:8 in a ventilation cabinet to prepare a stop solution.

Antigen coating:

adding 100 mu L coating solution (carbonate buffer solution with antigen concentration of 5 mu g/m L) into each well of a 96-well enzyme label plate, coating for 24h at 4 ℃, washing the plate 3 times by using an automatic plate washing machine, adding 250 mu L sealing solution into each well, sealing, incubating for 2h at 37 ℃, washing the plate 3 times by using the automatic plate washing machine, and storing in a refrigerator at 4 ℃ for later use.

Detection of serum-specific IgG and saliva, vaginal lavage fluid-specific IgA

To investigate the effect of three Acinetobacter baumannii OMVs on the humoral immune response level of the body, mice were immunized by intramuscular injection and nasal drip, mice serum was diluted at a ratio of 1:2000, samples were diluted at 200. mu. L per well and assayed 4 times in parallel, 100. mu. L antibody diluent was added to each well of the remaining columns, incubated at 37 ℃ for 1h, washed with an automatic plate washer for 3 times, HRP-labeled goat anti-mouse IgG antibody diluent (1:5000) 100. mu. L was added to each well, incubated at 37 ℃ for 40min, washed with an automatic plate washer for 3 times, 100. mu. L soluble monocomponent TMB substrate solution was added to each well, incubated at room temperature for 15min, stopped at 50. mu. L2M sulfuric acid solution per well, and OD of each well was measured with a 96-well plate reader_450nmThe value is obtained. Saliva, vaginal lavage specific IgA response levels were determined as above, with saliva and vaginal lavage diluted 1:4 and HRP-labeled goat anti-mouse IgA antibody used as the secondary antibody.

The detection results are shown in fig. 15-16, three acinetobacter baumannii OMVs can remarkably induce the mouse serum IgG antibody response through two immunization ways of intramuscular injection and nasal drip, and the serum IgG level of each group of mice gradually increases along with the immunization process. Of the three OMVs, SuOMV induced serum IgG levels were significantly higher than sOMV and nOMV. Furthermore, as shown in figures 17-20, suomvs and sOMV can also significantly increase the expression of specific sIgA in saliva and vaginal lavage fluid by nasal drip immunization, whereas immunization of three OMVs by the intramuscular route failed to induce a mucosal site sIgA response.

Detection of serum-specific IgG1, IgG2a

To further explore the types of specific cellular immune responses that may be enhanced by the OMVs of Acinetobacter baumannii, the expression levels of specific IgG1 and IgG2a in serum were determined.mouse serum was diluted at a ratio of 1:2000, 100. mu. L serum dilution was added to each well, and the assay was performed in parallel 4 times, incubation at 37 ℃ for 2h, 3 times, goat anti-mouse IgG2 ahevav chain (HRP) dilution (1:20000) or goat anti-mouse IgG1heavy chain (HRP) dilution (1:20000) 100. mu. L was added to each well, incubation at 37 ℃ for 40min, and the assay was performed 3 times using an automatic plate washer, 100. mu. L TMB substrate color developing solution was added to each well, and the reaction was stopped by shading at room temperature for 15min, 50. mu. L2M sulfuric acid solution was added to each well, and the OD of each well was measured using a 96-well microplate reader_450nmThe value is obtained.

The detection results are shown in fig. 21-22, SuOMV and sOMV induced significant IgG1 and IgG2a responses by the intramuscular immunization route, and SuOMV induced the highest IgG1 and IgG2a levels, and the response level of nOMV group was not significantly different from that of PBS group. In addition, by the nasal drip route, only SuOMV could significantly elevate the expression level of specific IgG1, although SuOMV and sOMV also induced a significant IgG2a response (fig. 23-24). From the ratio of IgG2a to IgG1 (IgG2a/IgG1), IgG2a/IgG1 was significantly higher in the myoglobin-immunized sOMV group than in the PBS group and other immune groups, and the ratio was greater than 1, suggesting that the myoglobin-immunized sOMV induces an antigen-specific Th1 biased cellular immune response (fig. 25); all nasal drops of the immunised groups IgG2a/IgG1 were greater than 1 (FIG. 26), suggesting that all three A.baumannii OMVs induced Th 1-biased cellular immune responses by nasal drops.

In conclusion, the method for extracting acinetobacter baumannii OMVs provided by the invention has the advantages that the yield of SuOMV extracted by a sucrose method is highest, three OMVs are spherical under a transmission electron microscope, wherein the average particle size of sOMV is 183.3nm, the average particle size of SuOMV is 142.9nm, the average particle size of nOMV is 269.9nm, protein components of the three OMVs have certain similarity but significant difference through SDS-PAGE and MA L DI-TOF MS analysis, the protein types with the highest content of the three OMVs are outer membrane proteins, and the three OMVs with the highest content of the single protein are OmpA, can significantly up-regulate the expression of CD40, CD80 and CD86 on the surface of BMDCs, which shows that the three OMVs can induce the maturation of BMDCs, and have the best effect, in addition, the three OMVs can stimulate the significant up-regulation of I L-1 and TNF- α in supernatant of BMDCs culture supernatant, and further show that the three OMVs can stimulate the significant up-11- β and TNF- α to induce the significant up- β and 5 Th-7 and 5 immune response of immune response to the nose-7 OMV, which is proved that the immune response of a sero is greater than the immune response of a T-S, the immune response of a T-S, a T-S, a T.

Further, the results of examples 1-2 to 1-3 were similar to those of example 1-1 but superior to those of examples 1-4 to 1-5, and the results of examples 2-2 to 2-3 were similar to those of example 2-1 but superior to those of examples 2-4 to 2-7, and the specific results were not shown for reasons of space.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. A method for extracting outer membrane vesicles from Acinetobacter baumannii, comprising: carrying out first solid-liquid separation on the fermentation liquor of the acinetobacter baumannii to obtain thallus sediment, and then adding a buffer solution without a detergent into the thallus sediment to destroy the cell walls of the thallus.

2. The method according to claim 1, wherein the cell wall of the microbial cells is disrupted by at least one method selected from the group consisting of enzymolysis, sonication, shearing, grinding, crushing, microfluidization, air-fortification, and osmotic shock.

3. The method according to claim 1, wherein the cell wall of the microbial cells is disrupted by shearing, and the detergent-free buffer comprises Tris-HCl, NaCl and EDTA;

preferably, the concentration of Tris-HCl in the buffer solution is 0.01-0.2M, NaCl, the concentration of Tris-HCl in the buffer solution is 0.05-0.3M, EDTA, the concentration of Tris-HCl in the buffer solution is 0.005-0.05M, and the pH value of the buffer solution is 6-9.

4. The method of claim 3, wherein the method of shearing comprises: adding the buffer solution into the thallus precipitate to obtain a thallus suspension, then carrying out incubation on the thallus suspension, and carrying out shearing dispersion on the thallus after the incubation so as to destroy the cell wall to obtain a wall-broken product;

preferably, the addition amount of the buffer solution is 1-10ml relative to 1g of the thallus precipitate;

preferably, the incubation conditions include: the temperature is 50-60 ℃, and the time is 20-40 min;

preferably, the conditions for shear dispersion include: the temperature is 20-40 ℃, the shearing speed is 8000-25000rpm, and the time is 1-10 min.

5. The method of claim 4, wherein the method further comprises: performing second solid-liquid separation on the wall-broken product to obtain a crude product of the acinetobacter baumannii outer membrane vesicles;

preferably, the second solid-liquid separation comprises a first centrifugation, a second centrifugation, a third centrifugation and a fourth centrifugation which are sequentially performed;

wherein the first centrifugation comprises: centrifuging the wall-broken product at 10000-;

the second centrifugation comprises: centrifuging the first supernatant at 20000-30000g and 2-6 ℃ to obtain a second supernatant;

the third centrifugation comprises: centrifuging the second supernatant at the temperature of 2-6 ℃ under the conditions of 30000-50000g to obtain a third supernatant;

the fourth centrifugation comprises: centrifuging the third supernatant at 50000-120000g and 2-6 ℃, and collecting precipitates to obtain a crude product of the acinetobacter baumannii outer membrane vesicles;

preferably, the method further comprises: washing the crude product of the acinetobacter baumannii outer membrane vesicle to obtain a pure product of the acinetobacter baumannii outer membrane vesicle;

preferably, the method of washing comprises: and (3) suspending the crude acinetobacter baumannii outer membrane vesicle in PBS buffer solution, performing centrifugal treatment under the conditions of 20000 plus 50000g and 2-6 ℃, and collecting precipitates to obtain a pure acinetobacter baumannii outer membrane vesicle.

6. The method according to claim 1, wherein the method for disrupting cell walls of the cells comprises an enzymatic method and an ultrasonic method, and the detergent-free buffer comprises sucrose, Tris-HCl, EDTA and lysozyme;

preferably, in the buffer solution, the concentration of sucrose is 0.05-1M, Tris-HCl is 0.01-0.2M, EDTA, the concentration of lysozyme is 0.05-20mg/ml, and the pH value of the buffer solution is 6-9;

preferably, the buffer is added in an amount of 15 to 100ml per 1g of the cell pellet.

7. The method according to claim 6, wherein the method for disrupting cell walls of the microbial cells by an enzymatic method and an ultrasonic method comprises: adding a first sucrose solution containing sucrose and EDTA into the thallus precipitate to obtain a heavy-suspension bacterium solution, then adding lysozyme and EDTA into the heavy-suspension bacterium solution, and performing ultrasonic wall breaking on the obtained mixed material;

preferably, the first sucrose solution is added in an amount of 15-100ml relative to 1g of the pellet, and the first sucrose solution contains 0.05-1M sucrose and 0.01-0.2M Tris-HCl;

preferably, the lysozyme and the EDTA are added in an amount to ensure that the concentration of the EDTA in the mixed material is 1-5mM, and the concentration of the lysozyme is 0.05-20 mg/ml;

preferably, the ultrasonic wall breaking is carried out for 5-15min under the ice bath condition, wherein each ultrasonic breaking is carried out for 3-5s, and the rest time is 4-6 s.

8. The method of claim 7, wherein the method further comprises: performing third solid-liquid separation on the wall-broken product to obtain a crude product of the acinetobacter baumannii outer membrane vesicles;

preferably, the third solid-liquid separation comprises a first centrifugation and a second centrifugation which are sequentially performed;

wherein the first centrifugation comprises: centrifuging the ultrasonic wall breaking product at the temperature of 2-6 ℃ under the conditions of 1000-2000g to obtain a first supernatant;

the second centrifugation comprises: centrifuging the first supernatant at 200000-500000g and 2-6 ℃, and collecting precipitates to obtain a crude product of acinetobacter baumannii outer membrane vesicles;

preferably, the method further comprises: resuspending the crude product of the acinetobacter baumannii outer membrane vesicle in a second sucrose solution, then performing sucrose density gradient centrifugation, and collecting a sucrose layer containing the acinetobacter baumannii outer membrane vesicle;

wherein the second sucrose solution contains 20-30 wt% sucrose and 4-6mM EDTA, and has a pH of 7-8;

the sucrose density gradient of the sucrose density gradient centrifugation is 30-55 wt%, and the sucrose layer containing the acinetobacter baumannii outer membrane vesicle is 45-50 wt% of the sucrose layer;

preferably, the sucrose density gradient centrifugation conditions comprise: the rotating speed is 200000-500000g, and the temperature is 2-6 ℃;

preferably, the method further comprises diluting the sucrose layer containing the acinetobacter baumannii outer membrane vesicles, then centrifuging at 40000-;

preferably, before resuspending the crude acinetobacter baumannii outer membrane vesicles in the second sucrose solution, the method further comprises: washing the crude product of the acinetobacter baumannii outer membrane vesicle in a third sucrose solution, and then re-suspending the washed solid phase in a second sucrose solution;

wherein the third sucrose solution contains 0.2-0.3M sucrose, 3-4mM Tris-HCl, 0.8-1.2mM EDTA, and has a pH value of 7.5-8;

preferably, the washed solid phase is obtained by centrifugation at 200000-.

9. An acinetobacter baumannii outer membrane vesicle prepared by the method of any one of claims 1 to 8.

10. Use of the method of any one of claims 1-8 or the acinetobacter baumannii outer membrane vesicle of claim 9 in the preparation of a medicament for the prevention and/or treatment of acinetobacter baumannii infection.