A method for improving the yield of Listeria monocytogenes membrane vesicles and its application

Abstract

The present invention relates to a method for improving the yield of Listeria monocytogenes membrane vesicles and its application. The LMΔdaldat strain with the dal and dat genes knocked out was constructed. Meanwhile, the complementation plasmid pCW633 carrying the dal gene was constructed and electrotransformed into the nutrient-deficient strain LMΔdaldat to construct the stable recombinant strain LMΔdaldat::pCW633. Compared with the wild strain LM, the yield of MVs secreted by the recombinant strain LMΔdaldat::pCW633 can be increased by 3.41 times, and the shape, structure, particle size and protein components of the secreted MVs have no obvious difference from those of the MVs secreted by the wild strain LM. Compared with the method of antibiotic treatment, the yield increase of MVs secreted by the recombinant strain LMΔdaldat::pCW633 constructed in the present invention is more significant.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for improving the production of Listeria monocytogenes membrane vesicles and its application. Background Art

[0002] Bacterial membrane vesicles (BMVs) are lipid bilayer membrane nanostructures released by bacteria into the extracellular environment during growth. Their diameters range from 20 to 200 nm and they contain various bacterial components, including lipids, proteins, nucleic acids, etc. They can mediate bacterial-bacterial and bacterial-host interactions and participate in various biological activities such as bacterial pathogenesis, signal transduction, quorum sensing, and stress response. BMVs can be divided into outer membrane vesicles (OMVs) secreted by Gram-negative bacteria (G - ) and membrane vesicles (MVs) secreted by Gram-positive bacteria (G + ). The secretion mechanisms of these two types of BMVs are different, and their structural components also vary greatly due to the different cell wall structures and compositions of the source bacteria. The complex and diverse components on BMVs endow them with good immunogenicity and can effectively stimulate the body to produce an immune response. The nanoscale vesicular structure also gives them the ability to carry foreign antigens and encapsulate small molecule drugs. Therefore, BMVs are considered a promising vaccine carrier platform or drug delivery system. The commercially available group B meningococcal vaccine MenBvac is based on Neisseria meningitidis OMVs, which also proves the broad application prospects of BMVs.

[0003] Currently, research on BMVs mainly focuses on OMVs secreted by Gram-negative bacteria, including OMVs secreted by Escherichia coli, Neisseria meningitidis, Pseudomonas aeruginosa, etc. However, it should be noted that OMVs contain lipopolysaccharide (LPS), a specific component of the G - -bacterial cell membrane. The presence of this pyrogen makes the safety of OMV-based biological agents questionable, which also limits the application of OMVs to a certain extent. MVs produced by G + -bacteria do not contain endotoxin LPS, so they have better safety. Therefore, biological agents developed based on G + -bacterial MVs have more advantages compared to OMVs. However, current research based on MVs is relatively scarce, and only a few G + -bacteria such as Staphylococcus aureus and Listeria monocytogenes have been reported. The main limiting factor lies in G+ The yield of bacterial MVs is relatively low. The secretion of MVs is related to the cross-linking degree of the peptidoglycan layer of the bacterial cell wall.

[0004] Listeria is a genus of Gram-positive + short bacilli without spores, which are intracellular parasites. Multiple species have been discovered so far, and the common ones include Listeria monocytogenes (LM) and Listeria ivanovii (LI). LM and LI can produce various virulence factors such as internalin and hemolysin, which promote phagocytosis and escape from lysosomes, and can simultaneously stimulate the body's cellular and humoral immune responses. In 2013, Lee et al. first discovered that LM can also produce MVs. The research found that LM MVs are similar to other MVs, having advantages such as good biosafety, high immunogenicity, and the ability to load exogenous antigens, but also having the problem of low yield. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method and application for improving the yield of Listeria monocytogenes membrane vesicles.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for improving the yield of Listeria monocytogenes membrane vesicles, and the specific steps are as follows:

[0008] (1) Knock out the dal and dat genes of the wild-type strain LM of Listeria monocytogenes to obtain the strain LMΔdaldat, and prepare the competent cells of LMΔdaldat;

[0009] (2) Electrotransform the targeting plasmid pCW633 into the competent cells of LMΔdaldat to obtain the strain LMΔdaldat::pCW633;

[0010] (3) Culture LMΔdaldat::pCW633, centrifuge to take the supernatant, and extract and purify to obtain Listeria monocytogenes membrane vesicles LMMVs.

[0011] As one of the preferred technical solutions, the specific method of step (1) is as follows:

[0012] (1-1) The wild strain LM of Listeria monocytogenes was used to prepare competent LM. The targeting plasmid pCW619-LMdal was electrotransformed into competent LM cells. After electrotransformation and recovery, the cells were cultured. Colony PCR screening was performed using the primer pCW619-Lmdal-F shown in SEQ ID NO.1 and the primer pCW619-R shown in SEQ ID NO.2 for homologous recombination. Colony PCR screening was then performed using the primer LMdal-F shown in SEQ ID NO.3, the primer LMdal-R shown in SEQ ID NO.4, the primer HomoLMdal-F shown in SEQ ID NO.5, and the primer HomoLMdal-R shown in SEQ ID NO.6 to obtain the strain LMΔdal. Competent cells of LMΔdal were prepared.

[0013] (1-2) The targeting plasmid pCW619-LMdat was electrotransformed into competent LMΔdal cells. After electrotransformation and recovery, the cells were cultured. Colony PCR screening was performed using the primer pCW619-Lmdat-F shown in SEQ ID NO.7 and the primer pCW619-R shown in SEQ ID NO.2 for homologous recombination. Colony PCR screening was then performed using the primer LMdat-F shown in SEQ ID NO.8, the primer LMdat-R shown in SEQ ID NO.9, the primer HomoLMdat-F shown in SEQ ID NO.10, and the primer HomoLMdat-R shown in SEQ ID NO.11 to obtain the strain LMΔdaldat. Competent cells of LMΔdaldat were prepared.

[0014] As one of the further preferred technical solutions, in step (1-1), the preparation method of the targeting plasmid pCW619-LMdal is as follows: The Escherichia coli strain carrying the pCW619-LMdal plasmid was streaked on an LB solid medium supplemented with 100 μg / mL ampicillin and cultured at 37 °C for 16 hours. A single colony was picked and inoculated into an LB liquid medium supplemented with 100 μg / mL ampicillin and cultured at 37 °C with shaking at 200 rpm for 16 hours to extract the plasmid pCW619-LMdal.

[0015] As one of the preferred technical solutions, in step (2), the preparation method of the strain LMΔdaldat::pCW633 is as follows: The targeting plasmid pCW633 was electrotransformed into competent LMΔdaldat cells. After electrotransformation and recovery, the cells were cultured. Colony PCR screening was performed using the primer asd-SX-F shown in SEQ ID NO.22 and the primer asd-SX-R shown in SEQ ID NO.23 to obtain the strain LMΔdaldat::pCW633.

[0016] As one of the further preferred technical solutions, the preparation method of the targeting plasmid pCW633 is as follows:

[0017] (2-A) Culture Escherichia coli carrying the plasmid pCW630, extract the plasmid pCW630, whose nucleotide sequence is shown in SEQ ID NO.12. Use the primer Vector633-F shown in SEQ ID NO.13 and the primer Vector633-R shown in SEQ ID NO.14 to amplify the vector fragment Vector633 from the plasmid pCW630, whose nucleotide sequence is shown in SEQ ID NO.17; Use the primer phly-LM dal-F shown in SEQ ID NO.15 and the primer phly-LM dal-R shown in SEQ ID NO.16 to amplify the inserted fragment phly-LM dal from the plasmid pCW630, whose nucleotide sequence is shown in SEQ ID NO.18. Connect the vector fragment Vector633 and the inserted fragment phly-LM dal to obtain a ligation product;

[0018] (2-B) Transform the ligation product into competent Escherichia coli DH5αΔasd cells, and perform colony PCR screening using the primer pCW633-SX-F shown in SEQ ID NO.19 and the primer pCW633-SX-R shown in SEQ ID NO.20, and extract the plasmid pCW633, whose nucleotide sequence is shown in SEQ ID NO.21.

[0019] As one of the preferred technical solutions, in step (3), the specific method for culturing LMΔdaldat::pCW633 is as follows: Streak the strain LMΔdaldat::pCW633 onto a BHI plate and culture it at 37°C for 24 hours; Pick a single colony on the plate and inoculate it into 5 mL of BHI broth, and incubate it at 37°C with shaking at 200 rpm for 16 hours; Pipette 250 μL of fresh bacterial solution and inoculate it into 25 mL of BHI broth, and incubate it at 37°C with shaking at 200 rpm for 16 hours; Adjust the OD600 value of the bacterial solution to 1.0, take the bacterial solution and inoculate it into BHI broth at a volume ratio of 1:100, and incubate it at 37°C with shaking at 200 rpm for 16 hours.

[0020] As one of the preferred technical solutions, in step (3), the process conditions for centrifugation are: Centrifuge at 4°C and 12000g for 10 minutes.

[0021] As one of the preferred technical solutions, in step (3), the specific method for extraction and purification is as follows:

[0022] (3-a) Filter the supernatant through a 0.22 μm filter membrane, ultrafiltrate and concentrate, ultracentrifuge to obtain a precipitate, and resuspend it with 0.01 mol / L PBS (pH 7.2) to obtain a crude extract product;

[0023] (3-b) The crude extract is subjected to ultra-high speed centrifugation using multi-layer iodixanol solutions with different concentrations, collected by layering, and the flagellum-free liquid layer is combined. The volume is made up with 0.01 mol / L PBS (pH 7.2). Ultra-high speed centrifugation is carried out again to obtain the precipitate, which is resuspended with 0.01 mol / L PBS (pH 7.2).

[0024] As one of the further preferred technical solutions, in step (3-a), the cut-off molecular weight for ultrafiltration concentration is 500 kDa, and the process conditions for ultra-high speed centrifugation are: 4 °C, centrifugation at 140,000 g for 6 hours.

[0025] As one of the further preferred technical solutions, in step (3-b), the mass concentrations of the iodixanol solutions are 45%, 35%, 30%, 25%, 20%, 15%, and 10%, which are layered into the centrifuge tube from bottom to top, and the crude extract is layered on the topmost layer.

[0026] As one of the further preferred technical solutions, in step (3-b), the process conditions for ultra-high speed centrifugation are: 4 °C, centrifugation at 180,000 g for 4 hours; the process conditions for the second ultra-high speed centrifugation are: 4 °C, centrifugation at 140,000 g for 6 hours.

[0027] The present invention also provides the application of the foregoing method in basic research related to LM MVs, specifically including applications in multi-omics research of LM MVs, research on potential biological functions, and preparation of preparations using LM MVs as carriers.

[0028] The beneficial effects of the present invention are as follows:

[0029] To solve the problem of the low yield of Listeria monocytogenes MVs, the present invention uses Listeria monocytogenes as the starting strain and increases the yield of LM MVs from the perspective of reducing the cross-linking degree of the peptidoglycan layer in the bacterial cell wall. During the synthesis of the bacterial cell wall, D-alanine (D-Ala) plays a crucial role in the cross-linking between peptidoglycan layers. The synthesis of D-Ala is related to the dal and dat genes. The dal gene controls the synthesis of alanine racemase (Alr), which can convert L-alanine into D-Ala, while the dat gene controls the synthesis of D-amino acid aminotransferase (D-AAT), which can generate D-Ala and α-ketoglutaric acid from D-glutamic acid and pyruvic acid through transamination. When the dal and dat genes on the bacterial genome are knocked out, the bacteria cannot form a cell wall and cannot grow in a medium without the addition of exogenous D-Ala. Therefore, in the present invention, the dal gene is added to the plasmid and complemented into the dal / dat deletion strain, so that on the premise of maintaining the growth of the bacteria, the cross-linking degree of the peptidoglycan layer in the bacterial cell wall can be reduced as much as possible, thereby achieving the purpose of increasing the yield of MVs. At present, there is no method for increasing the yield of MVs related to the dal and dat genes, so the present invention is original.

[0030] In the present invention, the LMΔdaldat strain with the dal and dat genes knocked out was constructed. Meanwhile, the complementary plasmid pCW633 carrying the dal gene was constructed and electrotransformed into the auxotrophic strain LMΔdaldat to construct the stable recombinant strain LMΔdaldat::pCW633. The MVs of the recombinant strain were collected by the LM MVs extraction (ultrafiltration concentration-ultracentrifugation method) and purification (density gradient centrifugation method) protocol in the present invention. Then, the total protein concentration of the recombinant MVs was determined using a BCA kit, and the morphology and particle size characteristics of the MVs were analyzed by transmission electron microscope (TEM) and dynamic light scatter (DLS). The protein composition on the MVs was analyzed by SDS-PAGE. Compared with the wild-type strain LM, the yield of MVs secreted by the recombinant strain LMΔdaldat::pCW633 can be increased by 3.41 times, and there is no obvious difference in the shape structure, particle size, and protein components of the MVs secreted by the recombinant strain and those secreted by the wild-type strain. After the LM strain was treated with antibiotics (ampicillin, gentamicin, erythromycin, trimethoprim), the MVs secreted by the strain were 1.47 times, 0.47 times, 1.36 times, and 0.85 times that of the LM group, respectively. Therefore, the increase in the yield of MVs secreted by the recombinant strain LMΔdaldat::pCW633 constructed in the present invention is more significant. In summary, the present invention can solve the problem of low yield of LM MVs and can be used for related basic and applied research on LM MVs.

[0031] The present invention can be used for basic research related to LM MVs, including multi-omics research on LM MVs (proteomics, lipidomics, metabolomics, etc.), research on the potential biological functions of LM MVs (pathogenicity, signal transduction, quorum sensing, stress response, etc.), and application of related preparations using LM MVs as carriers (drug delivery, antigen targeting, regulation of immune response, etc.). Brief Description of the Drawings

[0032] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail preferably with reference to the accompanying drawings, where:

[0033] Figure 1 It is a schematic diagram of the plasmids involved in the preparation of the recombinant strain LMΔdaldat::pCW633 in the present invention. Among them, a is the targeting plasmid pCW619-LMdal; b is the targeting plasmid pCW619-LMdat; c is the plasmid pCW630; d is the plasmid pCW633;

[0034] Figure 2 It is a schematic diagram of the construction process of the recombinant strain LMΔdaldat::pCW633 in the present invention;

[0035] Figure 3 Colony PCR verification results for the preparation of recombinant strain LMΔdaldat::pCW633 in the present invention. Among them, M is 250bp DNA Marker; in Figure a, 1 and 2 are the amplifications of LM and LMΔdaldat::pCW633 with primers HomoLMdal-F / R respectively; in Figure b, 1 and 2 are the amplifications of LM and LMΔdaldat::pCW633 with primers HomoLMdat-F / R respectively; in Figure c, 1 and 2 are the amplifications of plasmid pCW630 and Escherichia coli pCW633 with primers pCW633-SX-F / R respectively; in Figure d, 1 and 2 are the amplifications of plasmid pCW633 and LMΔdaldat::pCW633 with primers asd-SX-F / R respectively;

[0036] Figure 4 In vitro growth curves of wild strain LM and recombinant strain LMΔdaldat::pCW633 in the present invention;

[0037] Figure 5 Comparison of the yields of MVs secreted by wild strain LM and recombinant strain LMΔdaldat::pCW633 in the present invention, ** indicates P<0.01;

[0038] Figure 6 Transmission electron micrographs of MVs secreted by wild strain LM and recombinant strain LMΔdaldat::pCW633 in the present invention for observing the morphology of MVs; Figure a is the MVs secreted by wild strain LM; Figure b is the MVs secreted by recombinant strain LMΔdaldat::pCW633;

[0039] Figure 7 Dynamic light scattering analysis of MVs secreted by wild strain LM and recombinant strain LMΔdaldat::pCW633 in the present invention for characterizing the particle size distribution of MVs;

[0040] Figure 8 SDS-PAGE electrophoresis patterns of MVs secreted by wild strain LM and recombinant strain LMΔdaldat::pCW633 in the present invention for analyzing the protein components of MVs, M is the protein molecular weight marker; 1 is wild strain LM; 2 is recombinant strain LMΔdaldat::pCW633.

[0041] Figure 9 Comparison of the crude extract yields of MVs secreted by wild strain LM under different antibiotic treatment conditions. Detailed implementation manners

[0042] The following specific examples illustrate the implementation modes of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in the following examples only illustrate the basic concept of the present invention in a schematic manner. Without conflict, the following examples and the features in the examples can be combined with each other.

[0043] The Listeria monocytogenes used in this example is LM 10403s (Mahdy SE, Liu S, Su L, Zhang X, Chen H, Pei X, Wang C. Expression of the VP1 protein of FMDV integrated chromosomally with mutant Listeria monocytogenes strain induced both humoral and cellular immune responses. Appl Microbiol Biotechnol, 2019, 103(4): 1919-1929.). Listeria monocytogenes from other different sources is also applicable to this example.

[0044] Example 1. Construction of the LMΔdal strain

[0045] 1.1 Preparation of competent cells of wild-type LM

[0046] 1.1.1 Resuscitation of the bacterial strain

[0047] Take the LM bacterial strain stored at -20°C and streak it onto a Brain-Heart Infusion Broth (BHI) plate, and culture it at 37°C for 24 h. Pick a single colony and inoculate it onto another BHI plate, and culture it at 37°C for 24 h.

[0048] 1.1.2 Preparation of competent cells

[0049] Pick 3 - 4 single colonies from the above - mentioned plate and inoculate them into 15 mL of BHI broth (containing 0.5 mol / L sucrose), and incubate at 37 °C with shaking at 200 rpm for 16 hours. The next morning, inoculate the aforementioned 15 mL of fresh bacterial solution into 250 mL of BHI broth (containing 0.5 mol / L sucrose), and incubate at 37 °C with shaking at 200 rpm. Zero with BHI broth (containing 0.5 mol / L sucrose), and regularly monitor the OD600 value of the bacterial solution. When the OD600 value reaches 0.4, add penicillin G solution (final concentration is 12.5 μg / mL), and continue to incubate. When the OD600 value of the bacterial solution reaches 0.7 or starts to decline, collect the bacterial cells. Aliquot the bacterial solution into 50 mL centrifuge tubes, centrifuge at 13000 rpm at 4 °C for 5 min, and discard the supernatant. Add 20 mL of pre - cooled 0.5 mol / L sucrose solution to resuspend the bacterial cell pellet, centrifuge at 10000 rpm at 4 °C for 10 min, discard the supernatant, and repeat this step once. Resuspend the bacterial cell pellet with 300 μL of 0.5 mol / L sucrose solution, aliquot 50 μL per tube, and store at - 80 °C. The collection of bacterial cells should be carried out on ice throughout the process.

[0050] 1.2 Preparation of targeting plasmid pCW619 - LMdal (NCBI accession no. MN528127)

[0051] Streak - inoculate the Escherichia coli strain carrying the pCW619 - LMdal plasmid (Lei Y, Zhou Y, Zhang Y, Liu S, Tian S, Ou Q, Liu T, Huang H, Tang T, Wang C. A Listeria ivanovii balanced - lethal system may be a promising antigen carrier for vaccine construction. Microb Biotechnol. 2022 Nov; 15(11):2831 - 2844.) stored at - 20 °C onto an LB (Luria - Bertani) solid medium supplemented with 100 μg / mL ampicillin (Ampicillin, Amp) (hereinafter referred to as LA plate), and culture in an incubator at 37 °C for 16 hours. Pick a single colony and inoculate it into 5 mL of LB liquid medium supplemented with 100 μg / mL Amp (hereinafter referred to as LA broth), and incubate at 37 °C with shaking at 200 rpm for 16 hours. Extract the plasmid pCW619 - LMdal according to the instructions of the Omega plasmid extraction kit, and elute with an appropriate amount of sterile ddH2O. Measure the plasmid concentration and purity using Nanodrop 2000. The schematic diagram of the targeting plasmid pCW619 - LMdal is shown in Figure 1 a.

[0052] 1.3 Electroporation

[0053] Pre-cool 10 μL and 200 μL pipette tips and electroporation cuvettes at -20°C, and pre-heat 1000 μL pipette tips and BHI broth (containing 1 mol / L sucrose) at 37°C. Thaw LM competent cells and targeting plasmid pCW619-LMdal on ice. Pipette 5 μL of the plasmid solution dropwise into the corresponding 50 μL of competent cells, gently mix with fingertips, and incubate on ice for 5 min. Transfer the above mixture into the electroporation cuvette and incubate on ice for 5 min. Apply an electric shock at 1500 V for 5 ms on the electroporator, take out the electroporation cuvette, and incubate on ice for 5 min. Add 700 μL of BHI broth (containing 1 mol / L sucrose) to the electroporation cuvette, mix the bacteria, and then aspirate it into a 1.5 mL EP tube. Resuscitate at 37°C and 200 rpm for 2 h. After resuscitation, centrifuge at 12000 rpm for 2 min, discard about 600 μL of the supernatant, and resuspend the bacterial pellet. Spread all the resuspended bacterial solution onto BHI solid medium supplemented with 3 μg / mL erythromycin (Ery) (hereinafter referred to as BE3 plate), and culture in an incubator at 37°C for 48 h - 72 h.

[0054] 1.4 Screening after electroporation

[0055] Pick a single colony from the BE3 plate into 20 μL of ddH2O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. The screening primers are pCW619-Lmdal-F / pCW619-R. The sequence of pCW619-Lmdal-F is as shown in SEQ ID NO.1, and the sequence of pCW619-R is as shown in SEQ ID NO.2. Amplify according to the conventional PCR conditions. After amplification, analyze by agarose gel electrophoresis (1% agarose gel by mass concentration, 90 V voltage).

[0056] 1.5 Homologous recombination

[0057] Streak the single colony with successful electroporation on the D-BE3 plate, that is, BHI solid medium supplemented with 3 μg / mL erythromycin (Ery) and 200 μg / mL D-alanine (hereinafter referred to as D-BE3 plate), and culture in an incubator at 42°C for 3 consecutive passages. Scrape the bacterial lawn and inoculate it into BHI liquid medium supplemented with 200 μg / mL D-alanine (hereinafter referred to as D-BHI broth), and incubate at 30°C and 200 rpm for 16 h for 6 consecutive passages. Gradient dilute the 6th generation bacterial solution with 0.01 mol / L PBS (pH 7.2) solution to 10 -6 , take 100 μL of the diluted solution and spread it onto BHI solid medium supplemented with 200 μg / mL D-alanine (hereinafter referred to as D-BHI plate), and culture in an incubator at 37°C for 48 h.

[0058] 1.6 Screening

[0059] Pick single colonies on the above D-BHI plates and simultaneously streak-inoculate them on D-BE3 plates and D-BHI plates, and culture at 37°C for 24 h. Select single colonies that do not grow on the D-BE3 plates but grow on the D-BHI plates for colony PCR screening. Pick a little bit of bacterial lawn on the D-BHI plate into 20 μL of ddH2O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. The screening primers are LMdal-F / R primers and HomoLMdal-F / R primers (see a) in Figure 3 , and amplify according to the conventional PCR conditions. After the amplification is completed, analyze by agarose gel electrophoresis (1% agarose gel by mass concentration, 90 V voltage). The LMdal-F sequence is as shown in SEQ ID NO.3, the LMdal-R sequence is as shown in SEQ ID NO.4, the HomoLMdal-F sequence is as shown in SEQ ID NO.5, and the HomoLMdal-R sequence is as shown in SEQ ID NO.6.

[0060] 1.7 Sequencing

[0061] Send the amplification products of the HomoLMdal-F / R primers to the company for sequencing. The strains with correct sequencing are named LMΔdal, and the bacterial strains are preserved in time.

[0062] Example 2. Construction of the LMΔdaldat strain

[0063] 2.1 Preparation of LMΔdal competent cells

[0064] Refer to 1.1 in Example 1 to prepare LMΔdal competent cells. Note that the culture medium used in this step is D-BHI plates and D-BHI broth (containing 0.5 mol / L sucrose).

[0065] 2.2 Preparation of the targeting plasmid pCW619-LMdat (NCBI accession no. MN 528128)

[0066] Refer to the preparation of the targeting plasmid pCW619-LMdat in 1.2 of Example 1 (Lei Y, Zhou Y, Zhang Y, Liu S, Tian S, Ou Q, Liu T, Huang H, Tang T, Wang C. A Listeria ivanovii balanced-lethal system may be a promising antigen carrier for vaccine construction. Microb Biotechnol. 2022 Nov;15(11):2831-2844.). The schematic diagram of the plasmid is shown in Figure 1 b in

[0067] 2.3 Electroporation

[0068] Refer to 1.3 in Example 1 to electroporate the targeting plasmid pCW619-LMdat into LMΔdal competent cells. After electroporation and recovery, the bacterial suspension was spread on D-BE3 plates and cultured at 37 °C for 48 - 72 hours.

[0069] 2.4 Screening after electroporation

[0070] Refer to 1.4 in Example 1 to perform colony PCR screening on the single colonies growing on the D-BE3 plates. The screening primers are pCW619-Lmdat-F / pCW619-R, pCW619-Lmdat-F is as shown in SEQ ID NO.7, and the sequence of pCW619-R is as shown in SEQ ID NO.2.

[0071] 2.5 Homologous recombination

[0072] Refer to 1.5 in Example 1 to perform homologous recombination.

[0073] 2.6 Screening

[0074] Pick the single colonies on the above D-BHI plates and streak them on BHI plates, D-BE3 plates and D-BHI plates simultaneously, and culture them in a 37 °C incubator for 24 h. Select the single colonies that do not grow on the BHI plates and D-BE3 plates but grow on the D-BHI plates for colony PCR screening.

[0075] Pick a little bacterial lawn from the D-BHI plate into 20 μL of ddH2O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. The screening primers are LMdat-F / R and HomoLMdat-F / R (see the results of colony PCR verification in Figure 3b. The LMdat-F sequence is as shown in SEQ ID NO.8, the LMdat-R sequence is as shown in SEQ ID NO.9, the HomoLMdat-F sequence is as shown in SEQ ID NO.10, and the HomoLMdat-R sequence is as shown in SEQ ID NO.11.

[0076] 2.7 Sequencing

[0077] Send the amplification products of the HomoLMdat-F / R primers for sequencing by the company. The strains with correct sequencing results are named LMΔdaldat, and the bacterial strains are promptly preserved.

[0078] Example 3. Construction of the complementation plasmid pCW633

[0079] 3.1 Preparation of competent Escherichia coli DH5αΔasd (CaCl2 method)

[0080] 3.1.1 Resuscitation of bacterial strains

[0081] Pick the Escherichia coli DH5αΔasd bacterial strains preserved at -20°C and streak inoculate them onto a 5 mL LB solid medium supplemented with 50 μg / mL diaminopimelic acid (DAP) (hereinafter referred to as DAP-LB plate) and culture at 37°C for 16 hours.

[0082] 3.1.2 Preparation of competent Escherichia coli DH5αΔasd

[0083] Pick a single colony and inoculate it into 5 mL of LB liquid medium supplemented with 50 μg / mL DAP (hereinafter referred to as DAP-LB broth), and culture at 37°C with shaking at 200 rpm for 16 hours. Pipette 1 mL of fresh bacterial liquid and inoculate it into 50 mL of DAP-LB broth, and culture at 37°C with shaking at 200 rpm until the OD600 value is about 0.5, then collect the bacterial cells. Transfer the bacterial liquid to a 50 mL centrifuge tube, ice bath for 30 min, centrifuge at 8000 rpm at 4°C for 3 min, and discard the supernatant. Add 10 mL of pre-cooled 0.1 mol / L CaCl2 to resuspend, centrifuge at 6000 rpm at 4°C for 3 min, and discard the supernatant. Resuspend with 1 mL of pre-cooled 0.1 mol / L CaCl2, aliquot 50 μL per tube and store at -80°C.

[0084] 3.2 Preparation of vector fragments and insert fragments

[0085] 3.2.1 Extraction of plasmid pCW630

[0086] Pick the Escherichia coli strain carrying the pCW630 plasmid stored at -20°C and streak it onto an LB plate. Incubate at 37°C for 16 hours to revive the strain. Pick a single colony and inoculate it into 5 mL of LB broth. Incubate at 37°C with shaking at 200 rpm for 16 hours. Extract the plasmid pCW630 according to the instructions of the Omega plasmid extraction kit and elute it with 35 μL of sterile ddH2O. Measure the plasmid concentration and purity using a Nanodrop2000. The sequence of plasmid pCW630 is as shown in SEQ ID NO.12, and the plasmid schematic diagram is shown in Figure 1 c in

[0087] 3.2.2 Preparation of vector fragment and insert fragment

[0088] The primers Vector633-F / Vector633-R are used to amplify the vector fragment Vector633 from the plasmid pCW630, and the primers phly-LM dal-F / R are used to amplify the insert fragment phly-LM dal from the plasmid pCW630. Amplify according to the conventional PCR conditions. After the amplification, separate each fragment by agarose gel electrophoresis (1.5% agarose gel by mass concentration, 90 V voltage), and use the Omega E.Z.N.A. Gel Extraction Kit to cut the gel and purify and recover the fragments. The sequence of primer Vector633-F is as shown in SEQ ID NO.13, Vector633-R is as shown in SEQ ID NO.14, phly-LM dal-F is as shown in SEQ ID NO.15, phly-LM dal-R is as shown in SEQ ID NO.16, the sequence of the vector fragment Vector633 is as shown in SEQ ID NO.17, and the sequence of the insert fragment phly-LM dal is as shown in SEQ ID NO.18.

[0089] 3.3 Ligation and transformation

[0090] 3.3.1 Ligation

[0091] Use the ABclonal MultiF Seamless Assembly Mix (RK21020) seamless ligation kit to ligate the vector fragment Vector633 and the insert fragment phly-LM dal. See the instructions for specific operations.

[0092] 3.3.2 Transformation

[0093] Thaw competent cells of Escherichia coli DH5αΔasd on ice, add 5 μL of the ligation product, gently mix by fingertip, and incubate on ice for 30 min. Heat shock in a 42 °C water bath for 45 s, quickly transfer to ice, and incubate for 2 min. Add 700 μL of LB broth, and recover at 37 °C with 200 rpm for 1 h. After recovery, centrifuge at 12,000 rpm for 2 min, discard approximately 600 μL of the supernatant, and resuspend the cell pellet. Depending on the experimental needs, pipette different volumes of the resuspended bacterial suspension onto LB plates and culture at 37 °C for 16 h.

[0094] 3.4 Colony PCR

[0095] Pick a single colony grown on the LB plate into 20 μL of ddH2O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. Primers pCW633-SX-F / R are used for colony PCR screening (see c) in Figure 3 . The sequence of pCW633-SX-F is as shown in SEQ ID NO.19, and the sequence of pCW633-SX-R is as shown in SEQ ID NO.20.

[0096] 3.5 Sequencing

[0097] Select a single colony with correct colony PCR verification and inoculate it into LB broth. Extract the plasmid and send it to a sequencing company for sequencing. The correctly sequenced plasmid is named pCW633 and stored in Escherichia coli pCW633. The sequence of plasmid pCW633 is as shown in SEQ ID NO.21, and the plasmid schematic diagram is shown in Figure 1 d.

[0098] Example 4. Construction of LMΔdaldat::pCW633( Figure 2 )

[0099] 4.1 Preparation of LMΔdaldat Competent Cells

[0100] Refer to 1.1 in Example 1 to prepare LMΔdaldat competent cells, noting that the medium used in this step is D-BHI plates and D-BHI broth (containing 0.5 mol / L sucrose).

[0101] 4.2 Preparation of Complementary Plasmid pCW633

[0102] Streak the Escherichia coli strain pCW633 stored at -20 °C onto an LB plate and culture at 37 °C for 16 h to revive the strain. Pick a single colony and inoculate it into 5 mL of LB broth, and incubate at 37 °C with 200 rpm for 16 h. Operate according to the instructions of the Omega plasmid extraction kit to extract plasmid pCW633 and elute it with 35 μL of sterile ddH2O. Use Nanodrop 2000 to measure the plasmid concentration and purity.

[0103] 4.3 Electroporation

[0104] Refer to 1.3 in Example 1 to electroporate the targeting plasmid pCW633 into LMΔdaldat competent cells. After electroporation and recovery, the bacterial suspension was spread on BHI plates and cultured at 37°C for 48 - 72 h.

[0105] 4.4 Colony PCR

[0106] Refer to 1.4 in Example 1 to perform colony PCR screening on single colonies growing on BHI plates. The screening primers are asd - SX - F / R (see d) in Figure 3 for the verification results of colony PCR). The sequence of asd - SX - F is as SEQ ID NO.22, and the sequence of asd - SX - R is as SEQ ID NO.23.

[0107] 4.5 Strain preservation

[0108] The recombinant strains with correct colony PCR verification were promptly preserved and named LMΔdaldat::pCW633.

[0109] Example 5. Determination of the growth curve of LMΔdaldat::pCW633

[0110] Pick the recombinant strain LMΔdaldat::pCW633 preserved at - 20°C and streak - inoculate it on a BHI plate and culture at 37°C for 24 h. Pick a single colony and inoculate it into 5 mL of BHI broth, and incubate it at 37°C with shaking at 200 rpm for 16 hours. Take an appropriate amount of the bacterial solution and inoculate it into 50 mL of BHI broth, adjust the absorbance value OD600 at 600 nm to 0.06, incubate it at 37°C with shaking at 200 rpm, measure OD600 once every 1 h, and draw a growth curve based on OD600. The growth curves of LM and LMΔdaldat::pCW633 are as Figure 4 shown. The growth trends of LMΔdaldat::pCW633 and LM are basically the same.

[0111] Example 6. Extract, purify MVs and perform characterization analysis

[0112] 6.1 Large - volume preparation of culture supernatant

[0113] 6.1.1 Strain recovery

[0114] Pick the recombinant strain LMΔdaldat::pCW633 preserved at - 20°C and streak - inoculate it on a BHI plate and culture at 37°C for 24 h. Pick a single colony and inoculate it onto another BHI plate and culture it in an incubator at 37°C for 24 h.

[0115] 6.1.2 Large - volume preparation of culture supernatant

[0116] Pick a single colony on the above-mentioned BHI plate and inoculate it into 5 mL of BHI broth. Incubate it at 37 °C with shaking at 200 rpm for 16 hours. Pipette 250 μL of the fresh bacterial solution and inoculate it into 25 mL of BHI broth. Incubate it at 37 °C with shaking at 200 rpm for 16 h. After adjusting the OD600 value of the above bacterial solution to about 1.0, take 10 mL of the bacterial solution and inoculate it into 1 L of BHI broth. Incubate it at 37 °C with shaking at 200 rpm for 16 h. Centrifuge at 4 °C at 12000 g for 10 min to collect the culture supernatant.

[0117] 6.2 MVs extraction and purification

[0118] 6.2.1 Ultrafiltration concentration - ultracentrifugation

[0119] Filter the culture supernatant through a 0.22 μm filter membrane, and concentrate the filtrate to 35 mL through a 500 KDa cut-off module by ultrafiltration. Centrifuge the concentrated solution at 4 °C at 140000 g for 6 h, and discard the supernatant. Resuspend the precipitate with 500 μL of 0.01 mol / L PBS (pH 7.2) solution to obtain the crude MVs extract.

[0120] 6.2.2 Density gradient centrifugation for purification

[0121] Sequentially (from bottom to top) lay the iodixanol solutions with mass concentrations of 45%, 35%, 30%, 25%, 20%, 15%, and 10% in a 4 mL centrifuge tube. Lay the crude MVs extract on the top layer. Ultracentrifuge at 4 °C at 180000 g for 4 h (with the minimum acceleration for speed increase and decrease). After centrifugation, collect the samples layer by layer. Take 20 μL of each layer of the sample, add an appropriate amount of SDS-PAGE protein loading buffer, boil for 10 min to denature the protein, and observe the density gradient centrifugation effect by SDS-PAGE gel electrophoresis (5% stacking gel, 10% separating gel) and Coomassie brilliant blue staining.

[0122] 6.2.3 Washing and desalting

[0123] According to the Coomassie brilliant blue staining results, combine the flagella-free liquid layers, make up the volume with 0.01 mol / L PBS (pH 7.2) solution, and ultracentrifuge at 4 °C at 140000 g for 6 h again. Discard the supernatant. Resuspend the precipitate with 50 μL of 0.01 mol / L PBS (pH 7.2) solution to obtain the purified MVs product.

[0124] 6.3 MVs characterization

[0125] 6.3.1 Measuring protein concentration by BCA method

[0126] According to the instructions of the BCA protein assay kit, measure the protein concentration of the purified MVs product to characterize the yield of MVs. The results are as Figure 5As shown, the MVs production of LMΔdaldat::pCW633 was 164.30 μg / L culture supernatant, which was 3.41 times that of LM (48.17 μg / L culture supernatant).

[0127] 6.3.2 TEM Characterization of MVs Morphological Features

[0128] Drop the MVs suspension onto a 400-mesh copper grid, stain it using the 2% phosphotungstic acid negative staining method, observe it under a transmission electron microscope (80 KV), and characterize the morphological features of MVs. The MVs secreted by the wild-type strain LM are shown in Figure 6 a, and the MVs secreted by LMΔdaldat::pCW633 are shown in Figure 6 b. As Figure 6 shown, the MVs presented a typical spherical structure under TEM, with a clean background. Moreover, for the MVs secreted by LMΔdaldat::pCW633, there were no obvious differences in their shape, structure, size, etc. compared with those of LM.

[0129] 6.3.3 Dynamic Light Scattering Analysis for Characterizing MVs Particle Size Features

[0130] Dilute the MVs suspension to 1 mL with 0.01 mol / L PBS (pH 7.2) solution at a ratio of 1:20, add it to a cuvette and measure it on a machine (Malvern particle size analyzer, Zetasizer Nano ZS). Report the average particle size of MVs with the average particle size (Z-average), and show the particle size distribution characteristics of MVs with Intensity PSD. The results of the dynamic light scattering analysis are shown in Figure 7 . The average particle size of the MVs secreted by LM was 99.4 nm, and the average particle size of the MVs secreted by LMΔdaldat::pCW633 was 103.4 nm, and their particle size distributions were basically the same.

[0131] 6.3.4 SDS-PAGE for Characterizing MVs Protein Composition

[0132] Take 10 μL of the MVs sample, add an appropriate amount of SDS-PAGE protein loading buffer, and boil for 10 min to denature the protein. Prepare an SDS-PAGE gel (5% stacking gel, 10% separating gel), after loading, perform electrophoresis at a constant voltage of 80 V for 30 min and then transfer to 120 V for 1 h. After the electrophoresis is completed, stain it with Coomassie Brilliant Blue staining solution at room temperature for 1 h and then decolorize until the background is clean, and image it through a gel imaging system. As Figure 8 can be seen, for the MVs secreted by LMΔdaldat::pCW633, their protein composition components were the same as those of the MVs secreted by LM.

[0133] Example 7. MVs Secretion Amount under Different Antibiotic Treatment Conditions

[0134] 7.1 Large-volume preparation of culture supernatant

[0135] 7.1.1 Bacterial strain resuscitation

[0136] Pick the bacterial strain LM stored at -20°C and streak it onto a BHI plate, then culture it at 37°C for 24 h. Pick a single colony and inoculate it onto another BHI plate, then culture it in an incubator at 37°C for 24 h.

[0137] 7.1.2 Large-volume preparation of culture supernatant

[0138] Pick a single colony from the above BHI plate and inoculate it into 5 mL of BHI broth, then incubate it at 37°C with shaking at 200 rpm for 16 h. Pipette 250 μL of the fresh bacterial solution and inoculate it into 25 mL of BHI broth, then incubate it at 37°C with shaking at 200 rpm for 16 h. After adjusting the OD600 value of the above bacterial solution to about 1.0, take 10 mL of the bacterial solution and inoculate it into 1 L of BHI broth containing different antibiotics, then incubate it at 37°C with shaking at 200 rpm for 16 h. Centrifuge at 12000 g at 4°C for 10 min to collect the culture supernatant. Among them, the Ampicillin (Amp) group is BHI broth containing 0.1221 μg / mL Amp, the Gentamicin (Gen) group is BHI broth containing 0.4883 μg / mL Gen, the Erythromycin (Ery) group is BHI broth containing 0.0610 μg / mL Ery, and the Trimethoprim (Tmp) group is BHI broth containing 1.953 μg / mL Tmp.

[0139] 7.2 Extraction and characterization of MVs

[0140] 7.2.1 Ultrafiltration concentration - ultracentrifugation

[0141] Filter the culture supernatant through a 0.22 μm filter membrane, and concentrate the filtrate to 35 mL through a 500 KDa retention module. Ultracentrifuge the concentrated solution at 140000 g at 4°C for 6 h, and discard the supernatant. Resuspend the precipitate with 500 μL of 0.01 mol / L PBS (pH 7.2) solution to obtain the crude MVs product.

[0142] 7.2.2 Protein concentration determination by BCA method

[0143] According to the instructions of the BCA protein assay kit, determine the protein concentration of the crude MVs product to characterize the yield of MVs. The results are as Figure 9As shown, compared with the yield of LM MVs, the yield of MVs in the Amp treatment group was 1.47 times that of the LM group, the yield of MVs in the Ery treatment group was 1.36 times that of the LM group, while the yield of MVs in the Gen treatment group and the Tmp treatment group decreased compared with the LM group, being 0.47 times and 0.85 times that of the LM group, respectively.

[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the present technical solution, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for increasing the yield of Listeria monocytogenes membrane vesicles, characterized in that, The specific steps are as follows: (1) Knock out the dal and dat genes of the wild strain LM of Listeria monocytogenes, to obtain the strain LMΔ daldat , and prepare the competent cells of LMΔ daldat ; (2) Electrotransform the targeting plasmid pCW633 into LMΔ daldat competent cells to obtain the strain LMΔ daldat ::pCW633; (3) Culture LMΔ daldat ::pCW633, centrifuge to obtain the supernatant, and extract and purify to obtain Listeria monocytogenes membrane vesicles LMMVs; The preparation method of the targeting plasmid pCW633 is as follows: Culture Escherichia coli carrying plasmid pCW630, extract plasmid pCW630, the nucleotide sequence of which is shown in SEQ ID NO.12, and amplify the vector fragment from plasmid pCW630 using primer Vector633-F shown in SEQ ID NO.13 and primer Vector633-R shown in SEQ ID NO.14 Vector633 , the nucleotide sequence of which is shown in SEQ ID NO.17; amplify the insert fragment from plasmid pCW630 using primer phly-LM dal-F shown in SEQ ID NO.15 and primer phly-LM dal-R shown in SEQ ID NO.16 phly-LM dal , the nucleotide sequence of which is shown in SEQ ID NO.18, and ligate the vector fragment Vector633 and the insert fragment phly-LM dal to obtain a ligation product; Transformation of Escherichia coli DH5αΔ with the (2-B) ligation product asd Competent cells were used, and colony PCR screening was performed using the primer pCW633-SX-F shown in SEQ ID NO.19 and the primer pCW633-SX-R shown in SEQ ID NO.

20. The plasmid pCW633 was extracted, and its nucleotide sequence is shown in SEQ ID NO.

21.

2. The method according to claim 1, characterized in that The specific method of step (1) is as follows: (1-1) The wild strain LM of Listeria monocytogenes was used to prepare competent LM, and the targeting plasmid pCW619-LM dal was electrotransformed into competent LM cells. After electrotransformation and recovery, the cells were cultured. Colony PCR screening was performed using the primer pCW619-Lmdal-F shown in SEQ ID NO.1 and the primer pCW619-R shown in SEQ ID NO.2 for homologous recombination. Colony PCR screening was then performed using the primer LMdal-F shown in SEQ ID NO.3, the primer LMdal-R shown in SEQ ID NO.4, the primer HomoLMdal-F shown in SEQ ID NO.5, and the primer HomoLMdal-R shown in SEQ ID NO.6 to obtain the strain LMΔ dal , and competent cells of LMΔ dal were prepared; (1-2) The targeting plasmid pCW619-LM dat was electrotransformed into LMΔ dal competent cells. After electrotransformation and recovery, the cells were cultured. Colony PCR screening was performed using the primer pCW619-Lmdat-F shown in SEQ ID NO.7 and the primer pCW619-R shown in SEQ ID NO.2 for homologous recombination. Colony PCR screening was then performed using the primer LMdat-F shown in SEQ ID NO.8, the primer LMdat-R shown in SEQ ID NO.9, the primer HomoLMdat-F shown in SEQ ID NO.10, and the primer HomoLMdat-R shown in SEQ ID NO.11 to obtain the strain LMΔ daldat , and competent cells of LMΔ daldat were prepared.

3. The method according to claim 1, characterized in that, In step (2), the preparation method of strain LMΔ daldat ::pCW633 is as follows: The targeting plasmid pCW633 is electrotransformed into LMΔ daldat competent cells. After electrotransformation and resuscitation, the cells are cultured, and colony PCR screening is performed using the primer asd-SX-F shown in SEQ ID NO.22 and the primer asd-SX-R shown in SEQ ID NO.23 to obtain strain LMΔ daldat ::pCW633.

4. The method according to claim 1, wherein In step (3), LMΔ daldat The specific method for culturing ::pCW633 is as follows: Streak the strain LMΔ daldat ::pCW633 onto a BHI plate and culture it at 37°C for 24 hours; Pick a single colony from the plate and inoculate it into 5 mL of BHI broth, and incubate it at 37°C with shaking at 200 rpm for 16 hours; Pipette 250 μL of the fresh bacterial solution and inoculate it into 25 mL of BHI broth, and incubate it at 37°C with shaking at 200 rpm for 16 hours; Adjust the OD600 value of the bacterial solution to 1.0, take the bacterial solution and inoculate it into BHI broth at a volume ratio of 1:100, and incubate it at 37°C with shaking at 200 rpm for 16 hours.

5. The method according to claim 1, characterized in that In step (3), the process conditions for centrifugation are: centrifugation at 4°C and 12,000 g for 10 minutes.

6. The method according to claim 1, wherein In step (3), the specific method for extraction and purification is as follows: (3-a) Filter the supernatant through a 0.22 μm filter membrane, ultrafiltration and concentration, ultra-high speed centrifugation to obtain the precipitate, and resuspend it with 0.01 mol / L PBS to obtain the crude extract; (3-b) Ultra-high speed centrifuge the crude extract with iodixanol solutions of different concentrations in multiple layers, collect the layers separately, combine the layer without flagella, make up the volume with 0.01 mol / L PBS, ultra-high speed centrifuge again to obtain the precipitate, and resuspend it with 0.01 mol / L PBS to obtain the product.

7. The method according to claim 6, wherein In step (3-a), the cut-off molecular weight for ultrafiltration and concentration is 500 kDa, and the process conditions for ultra-high speed centrifugation are: centrifugation at 4°C and 140,000 g for 6 hours.

8. The method according to claim 6, characterized in that In step (3-b), the mass concentrations of the iodixanol solutions are 45%, 35%, 30%, 25%, 20%, 15%, and 10%, which are layered into the centrifuge tube from bottom to top, and the crude extract is layered on the top layer; The process conditions for ultra-high speed centrifugation are: centrifugation at 4°C and 180,000 g for 4 hours; the process conditions for ultra-high speed centrifugation again are: centrifugation at 4°C and 140,000 g for 6 hours.

9. Use of the method according to any one of claims 1 to 8 in the preparation of a preparation using LM MVs as a carrier.

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