A method for improving the production of Listeria monocytogenes membrane vesicles in sheep and its application

Abstract

The present invention relates to a method for improving the yield of Listeria monocytogenes (LI) membrane vesicles and its application. The LIΔdaldat 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 nutrient-deficient strain LIΔdaldat to construct the stable recombinant strain LIΔdaldat::pCW633. Compared with the wild-type strain LI, the yield of MVs secreted by the recombinant strain LIΔdaldat::pCW633 can be increased by 1.44 times, and the shape, structure, particle size and protein components of the MVs secreted by the recombinant strain have no obvious differences from those of the MVs secreted by the wild-type strain. The present invention can solve the problem of low yield of LI MVs and can be used for basic research related to LI MVs, including multi-omics research of LI MVs, research on potential biological functions of LI MVs, and application of related preparations using LI MVs as carriers.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for improving the yield of Listeria monocytogenes membrane vesicles in sheep 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 unique 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. The 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 degree of cross-linking of the peptidoglycan layer of the bacterial cell wall.

[0004] Listeria is a type of Gram + negative, non-spore-forming bacilli that 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. The applicant found that the MVs produced by LI are similar to other MVs, with good biosafety, high immunogenicity, and the ability to load foreign antigens, etc., but also have 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 for improving the yield of Listeria ivanovii membrane vesicles and its application.

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

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

[0008] (1) Knock out the dal and dat genes of the wild strain LI of Listeria ivanovii to obtain the strain LIΔdaldat, and prepare competent cells of LIΔdaldat;

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

[0010] (3) Culture LIΔdaldat::pCW633, centrifuge to take the supernatant, and extract and purify to obtain Listeria ivanovii membrane vesicles LIMVs.

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

[0012] (1-1) The competent LI was prepared from the wild strain LI of Listeria monocytogenes. The targeting plasmid pCW619-LIdal was electrotransformed into the competent LI cells. After electrotransformation and recovery, the cells were cultured. Colony PCR screening was performed using the primer pCW619-LIdal-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 LIdal-F shown in SEQ ID NO.3, the primer LIdal-R shown in SEQ ID NO.4, the primer HomoLIdal-F shown in SEQ ID NO.5, and the primer HomoLIdal-R shown in SEQ ID NO.6 to obtain the strain LIΔdal. The competent cells of LIΔdal were prepared.

[0013] (1-2) The targeting plasmid pCW619-LIdat was electrotransformed into the competent LIΔdal cells. After electrotransformation and recovery, the cells were cultured. Colony PCR screening was performed using the primer pCW619-LIdat-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 LIdat-F shown in SEQ ID NO.8, the primer LIdat-R shown in SEQ ID NO.9, the primer HomoLIdat-F shown in SEQ ID NO.10, and the primer HomoLIdat-R shown in SEQ ID NO.11 to obtain the strain LIΔdaldat. The competent cells of LIΔdaldat were prepared.

[0014] As one of the further preferred technical solutions, in step (1-1), the preparation method of the targeting plasmid pCW619-LIdal is as follows: The pCW619-LIdal Escherichia coli strain 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-LIdal.

[0015] As one of the preferred technical solutions, in step (2), the preparation method of the strain LIΔdaldat::pCW633 is as follows: The targeting plasmid pCW633 was electrotransformed into the competent LIΔdaldat 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 LIΔdaldat::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, the nucleotide sequence of which 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, the nucleotide sequence of which 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 insert fragment phly-LM dal from the plasmid pCW630, the nucleotide sequence of which is shown in SEQ ID NO.18. Connect the vector fragment Vector633 and the insert fragment phly-LM dal to obtain a ligation product.

[0018] (2-B) Transform the ligation product into Escherichia coli DH5αΔasd competent 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. Extract the plasmid pCW633, the nucleotide sequence of which is shown in SEQ ID NO.21.

[0019] As one of the preferred technical solutions, in step (3), the specific method for culturing LIΔdaldat::pCW633 is as follows: Streak the strain LIΔdaldat::pCW633 on a BHI plate and culture it at 37°C for 24 hours. 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. Pipette 250 μL of the 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 it, centrifuge at ultra-high speed to obtain a precipitate, and resuspend it with 0.01 mol / L PBS (pH 7.2) to obtain a crude extract.

[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), and then 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 LI MVs, specifically including applications in multi-omics research of LI MVs, research on potential biological functions, and preparation of preparations using LI 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 in sheep, the present invention increases the yield of LI 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, the present invention adds the dal gene to the plasmid and complements it into the dal and dat deletion strains, so as to reduce the cross-linking degree of the peptidoglycan layer in the bacterial cell wall as much as possible on the premise of maintaining the growth of the bacteria, thereby achieving the purpose of increasing the yield of MVs. At present, there is no method for improving the yield of MVs related to the dal and dat genes, so the present invention is innovative.

[0030] The present invention constructs the LIΔdaldat strain with the dal and dat genes knocked out, and simultaneously constructs the complementation plasmid pCW633 carrying the dal gene, and electrotransforms it into the auxotrophic strain LIΔdaldat to construct a stable recombinant strain LIΔdaldat::pCW633. The MVs of the recombinant strain are collected through the LI MVs extraction (ultrafiltration concentration-ultracentrifugation method) and purification (density gradient centrifugation method) protocols established and optimized by the applicant. Then, the total protein concentration of the recombinant MVs is measured using a BCA kit, and the morphology and particle size characteristics of the MVs are analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the protein composition on the MVs is analyzed by SDS-PAGE. Compared with the wild strain LI, the yield of MVs secreted by the recombinant strain LIΔdaldat::pCW633 can be increased by 1.44 times, and the shape structure, particle size and protein components of the MVs secreted by the recombinant strain have no obvious difference from those of the MVs secreted by the wild strain.

[0031] In summary, the present invention can solve the problem of low production of LI MVs and can be used in basic research related to LI MVs, including multi-omics research of LI MVs (proteomics, lipidomics, metabolomics, etc.), research on potential biological functions of LI MVs (pathogenesis, signal transduction, quorum sensing, stress response, etc.), and application of related preparations using LI 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 and preferably below in conjunction with the accompanying drawings, where:

[0033] Figure 1 It is a schematic diagram of the plasmids involved in the preparation of the recombinant strain LIΔdaldat::pCW633 of the present invention. Among them, a is the targeting plasmid pCW619-LIdal; b is the targeting plasmid pCW619-LIdat; 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 LIΔdaldat::pCW633 of the present invention;

[0035] Figure 3 It is the colony PCR verification result of the recombinant strain LIΔdaldat::pCW633 prepared in the present invention. Among them, M is the 250bp DNA Marker; in Figure a, 1 and 2 are the amplifications of LI and LIΔdaldat::pCW633 respectively with the primers HomoLIdal-F / R; in Figure b, 1 and 2 are the amplifications of LI and LIΔdaldat::pCW633 respectively with the primers HomoLIdat-F / R; in Figure c, 1 and 2 are the amplifications of the plasmid pCW630 and Escherichia coli pCW633 respectively with the primers pCW633-SX-F / R; in Figure d, 1 and 2 are the amplifications of the plasmid pCW633 and LIΔdaldat::pCW633 respectively with the primers asd-SX-F / R;

[0036] Figure 4 It is the in vitro growth curve of the wild strain LI and the recombinant strain LIΔdaldat::pCW633 in the present invention;

[0037] Figure 5 It is the comparison of the yields of MVs secreted by the wild strain LI and the recombinant strain LIΔdaldat::pCW633 in the present invention;

[0038] Figure 6This is a transmission electron micrograph of MVs secreted by the wild-type strain LI and the recombinant strain LIΔdaldat::pCW633 in the present invention, used to observe the morphology of MVs; a shows the MVs secreted by LI; b shows the MVs secreted by LIΔdaldat::pCW633.

[0039] Figure 7 This is the dynamic light scattering analysis of MVs secreted by the wild-type strain LI and the recombinant strain LIΔdaldat::pCW633 in the present invention, used to characterize the particle size distribution of MVs;

[0040] Figure 8 This is the SDS-PAGE electrophoresis pattern of MVs secreted by the wild-type strain LI and the recombinant strain LIΔdaldat::pCW633 in the present invention, used to analyze the protein components of MVs. M is the protein molecular weight marker; 1 is the wild-type strain LI; 2 is the recombinant strain LIΔdaldat::pCW633. Detailed implementation manners

[0041] The following specific examples illustrate the implementation manners 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 manners. Various 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.

[0042] The Listeria ivanovii used in this example is LI PAM55 (Liang Q, Li R, Liu S, Zhang Y, Tian S, Ou Q, Chen Z, Wang C. Recombinant Listeria ivanovii strain expressing listeriolysin O in place of ivanolysin O might be a potential antigen carrier for vaccine construction. Front Microbiol. 2022 Jul 22; 13: 962326.). Other Listeria ivanovii from different sources are also applicable to this example.

[0043] Example 1. Construction of the LIΔdal strain

[0044] 1.1 Preparation of competent cells of the wild-type strain LI

[0045] 1.1.1 Resuscitation of Bacterial Strains

[0046] Inoculate the LI bacterial strain stored at -20°C onto a Brain-Heart Infusion Broth (BHI) plate by streaking, 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.

[0047] 1.1.2 Preparation of Competent Cells

[0048] Pick 3 - 4 single colonies from the above 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 h. 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 begins 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 needs to be carried out on ice throughout the process.

[0049] 1.2 Preparation of Targeting Plasmid pCW619-LIdal (NCBI accession no. MN528129)

[0050] Pick the Escherichia coli strain carrying pCW619-LIdal (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 maybe a promising antigen carrier for vaccine construction. Microb Biotechnol. 2022 Nov;15(11):2831-2844.) stored at -20°C and streak it onto an LB (Luria-Bertani) solid medium supplemented with 100 μg / mL ampicillin (Amp) (hereinafter referred to as LA plate), and culture it 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 it at 37°C with shaking at 200 rpm for 16 hours. Extract the plasmid pCW619-LIdal according to the instructions of the Omega plasmid extraction kit, and elute it with an appropriate amount of sterile ddH 2 O. Measure the plasmid concentration and purity using Nanodrop2000. The schematic diagram of the targeting plasmid pCW619-LIdal is shown in Figure 1 a in

[0051] 1.3 Electroporation

[0052] 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 the LI competent cells and the targeting plasmid pCW619-LIdal 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 to an electroporation cuvette and incubate on ice for 5 min. Apply a voltage of 1500 V on the electroporator for 5 ms, 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 with shaking at 200 rpm for 2 h. After the resuscitation, centrifuge at 12000 rpm for 2 min, discard about 600 μL of the supernatant, and resuspend the bacterial pellet. Spread all of the resuspended bacterial solution onto a BHI solid medium supplemented with 3 μg / mL erythromycin (Ery) (hereinafter referred to as BE 3 plate), and culture it in an incubator at 37°C for 48 h - 72 h.

[0053] 1.4 Screening after electroporation

[0054] Pick BE3 A single colony on the plate was transferred to 20 μL of ddH 2 O to prepare a bacterial suspension. Using this bacterial suspension as a template, colony PCR screening was performed. The screening primers were pCW619-LIdal-F / pCW619-R. The sequence of pCW619-LIdal-F was as shown in SEQ ID NO.1, and the sequence of pCW619-R was as shown in SEQ ID NO.2. Amplification was carried out under conventional PCR conditions. After amplification, analysis was performed by agarose gel electrophoresis (1% (mass concentration) agarose gel, 90 V voltage).

[0055] 1.5 Homologous recombination

[0056] The single colony with successful electroporation was streaked on a D-BE 3 plate, namely, a BHI solid medium supplemented with 3 μg / mL erythromycin (Erythromycin, Ery) and 200 μg / mL D-alanine (hereinafter referred to as the D-BE 3 plate), and cultured in an incubator at 42 °C for 3 consecutive passages. The bacterial lawn was scraped and inoculated into a BHI liquid medium supplemented with 200 μg / mL D-alanine (hereinafter referred to as D-BHI broth), and incubated at 30 °C and 200 rpm for 16 hours for 6 consecutive passages. The 6th generation of the bacterial solution was serially diluted with 0.01 mol / L PBS (pH 7.2) solution to 10 -6 , and 100 μL of the diluted solution was spread on a BHI solid medium supplemented with 200 μg / mL D-alanine (hereinafter referred to as the D-BHI plate), and cultured in an incubator at 37 °C for 48 h.

[0057] 1.6 Screening

[0058] Single colonies on the above-mentioned D-BHI plate were picked and streaked simultaneously on a D-BE 3 plate and a D-BHI plate and cultured at 37 °C for 24 h. Single colonies that did not grow on the D-BE 3 plate and grew on the D-BHI plate were selected for colony PCR screening. A little bacterial lawn on the D-BHI plate was picked and transferred to 20 μL of ddH 2 O to prepare a bacterial suspension. Using this bacterial suspension as a template, colony PCR screening was performed. The screening primers were LIdal-F / R primers and HomoLIdal-F / R primers (for the results of colony PCR verification, see Figure 3In a), it can be amplified under conventional PCR conditions. After the amplification is completed, it is analyzed by agarose gel electrophoresis (1% agarose gel by mass concentration, 90 V voltage). The LIdal-F sequence is as shown in SEQ ID NO.3, the LIdal-R sequence is as shown in SEQ ID NO.4, the HomoLIdal-F sequence is as shown in SEQ ID NO.5, and the HomoLIdal-R sequence is as shown in SEQ ID NO.6.

[0059] 1.7 Sequencing

[0060] Send the amplification product of the HomoLIdal-F / R primer to the company for sequencing. The strains with correct sequencing are named LIΔdal, and the bacterial strains are preserved in time.

[0061] Example 2. Construction of the LIΔdaldat strain

[0062] 2.1 Preparation of LIΔdal competent cells

[0063] Refer to 1.1 in Example 1 to prepare LIΔdal competent cells. Note that the culture medium used in this step is D-BHI plate and D-BHI broth (containing 0.5 mol / L sucrose).

[0064] 2.2 Preparation of the targeting plasmid pCW619-LIdat (NCBI accession no. MN 528130)

[0065] Refer to 1.2 in Example 1 to prepare the targeting plasmid pCW619-LIdat (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 plasmid schematic diagram is shown in Figure 1 b in.

[0066] 2.3 Electroporation

[0067] Refer to 1.3 in Example 1 to electroporate the targeting plasmid pCW619-LIdat into LIΔdal competent cells. After electroporation and recovery, spread the bacterial suspension on the D-BE 3 plate and culture it at 37 °C for 48 - 72 h.

[0068] 2.4 Screening after electroporation

[0069] Refer to 1.4 in Example 1 for D-BE 3 Perform colony PCR screening on single colonies grown on the flat plate. The screening primers are pCW619-LIdat-F / pCW619-R. pCW619-LIdat-F is as shown in SEQ ID NO.7, and the sequence of pCW619-R is as shown in SEQ ID NO.2.

[0070] 2.5 Homologous recombination

[0071] Perform homologous recombination with reference to 1.5 in Example 1.

[0072] 2.6 Screening

[0073] Pick single colonies on the above D-BHI flat plate and streak them onto the BHI flat plate, D-BE 3 flat plate and D-BHI flat plate, and culture them in an incubator at 37°C for 24 h. Select single colonies that do not grow on the BHI flat plate and D-BE 3 flat plate but grow on the D-BHI flat plate for colony PCR screening.

[0074] Pick a little bacterial lawn from the D-BHI flat plate into 20 μL ddH 2 O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. The screening primers are LIdat-F / R and HomoLIdat-F / R (see the results of colony PCR verification in Figure 3 b). The sequence of LIdat-F is as shown in SEQ ID NO.8, the sequence of LIdat-R is as shown in SEQ ID NO.9, the sequence of HomoLIdat-F is as shown in SEQ ID NO.10, and the sequence of HomoLIdat-R is as shown in SEQ ID NO.11.

[0075] 2.7 Sequencing

[0076] Send the amplification products of the HomoLIdat-F / R primers to the company for sequencing. The strains with correct sequencing are named LIΔdaldat, and the bacterial strains are saved in time.

[0077] Example 3. Construct the complementary plasmid pCW633

[0078] 3.1 Prepare competent Escherichia coli DH5αΔasd (CaCl 2 method)

[0079] 3.1.1 Resuscitation of bacterial strains

[0080] Pick the Escherichia coli DH5αΔasd strain stored at -20°C and streak it onto a 5 mL LB solid medium supplemented with 50 μg / mL diaminopimelic acid (DAP) (hereinafter referred to as DAP-LB plate), and culture it at 37°C for 16 hours.

[0081] 3.1.2 Preparation of competent Escherichia coli DH5αΔasd

[0082] 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 incubate it at 37°C with shaking at 200 rpm for 16 hours. Pipette 1 mL of fresh bacterial solution and inoculate it into 50 mL of DAP-LB broth, and incubate it at 37°C with shaking at 200 rpm until the OD600 value is about 0.5, then collect the bacteria. Transfer the bacterial solution 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 CaCl 2 Resuspend, centrifuge at 6000 rpm at 4°C for 3 min, and discard the supernatant. Use 1 mL of pre-cooled 0.1 mol / L CaCl 2 Resuspend, aliquot 50 μL per tube and store at -80°C.

[0083] 3.2 Preparation of vector fragment and insert fragment

[0084] 3.2.1 Extraction of plasmid pCW630

[0085] Pick the Escherichia coli strain carrying the pCW630 plasmid stored at -20°C and streak it onto an LB plate to culture the strain at 37°C for 16 hours for resuscitation. Pick a single colony and inoculate it into 5 mL of LB broth, and incubate it at 37°C with shaking at 200 rpm for 16 hours. Operate according to the instructions of the Omega plasmid extraction kit to extract plasmid pCW630, and elute it with 35 μL of sterile ddH2O. Use Nanodrop2000 to measure the plasmid concentration and purity. 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 the figure.

[0086] 3.2.2 Preparation of vector fragment and insert fragment

[0087] 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 inserted fragment phly-LM dal from the plasmid pCW630. Amplification can be carried out under conventional PCR conditions. After the amplification is completed, each fragment is separated by agarose gel electrophoresis (1.5% agarose gel for plasmid concentration, 90V voltage), and the fragments are recovered by gel extraction and purification using the Omega E.Z.N.A. Gel Extraction Kit. The sequence of primer Vector633-F is as shown in SEQ ID NO.13, the sequence of Vector633-R is as shown in SEQ ID NO.14, the sequence of phly-LM dal-F is as shown in SEQ ID NO.15, the sequence of 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 inserted fragment phly-LM dal is as shown in SEQ ID NO.18.

[0088] 3.3 Ligation and Transformation

[0089] 3.3.1 Ligation

[0090] Use the ABclonal MultiF Seamless Assembly Mix (RK21020) seamless ligation kit to ligate the vector fragment Vector633 and the inserted fragment phly-LM dal. See the instruction manual for specific operations.

[0091] 3.3.2 Transformation

[0092] Thaw the competent cells of Escherichia coli DH5αΔasd on ice, add 5 μL of the ligation product, gently mix by fingertip, and let it stand on ice for 30 min. Heat shock at 42 °C in a water bath for 45 s, quickly transfer to ice, and let it stand for 2 min. Add 700 μL of LB broth, and resuscitate at 37 °C and 200 rpm for 1 h. After the resuscitation is completed, centrifuge at 12000 rpm for 2 min, discard about 600 μL of the supernatant, and resuspend the cell pellet. According to the experimental needs, pipette different volumes of the resuspended bacterial solution and spread it on an LB plate for culturing at 37 °C for 16 hours.

[0093] 3.4 Colony PCR

[0094] Pick a single colony grown on the LB plate into 20 μL of ddH 2 O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. The primers pCW633-SX-F / R are used for colony PCR screening (see the results of colony PCR verification in Figure 3In c). The pCW633-SX-F sequence is as shown in SEQ ID NO.19, and the pCW633-SX-R sequence is as shown in SEQ ID NO.20.

[0095] 3.5 Sequencing

[0096] Single colonies with correct colony PCR verification were selected and inoculated into LB broth, and the plasmids were extracted and sent to a sequencing company for sequencing. The plasmids with correct sequencing were 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 In d.

[0097] Example 4. Construction of LIΔdaldat::pCW633 ( Figure 2 )

[0098] 4.1 Preparation of LIΔdaldat competent cells

[0099] Refer to 1.1 in Example 1 to prepare LIΔdaldat competent cells. Note that the culture media used in this step are D-BHI plates and D-BHI broth (containing 0.5 mol / L sucrose).

[0100] 4.2 Preparation of the complementation plasmid pCW633

[0101] The Escherichia coli strain pCW633 stored at -20°C was streaked on an LB plate and cultured at 37°C for 16 hours to revive the strain. A single colony was picked and inoculated into 5 mL of LB broth, and cultured at 37°C with shaking at 200 rpm for 16 hours. According to the instructions of the Omega plasmid extraction kit, plasmid pCW633 was extracted and eluted with 35 μL of sterile ddH2O. The plasmid concentration and purity were measured using Nanodrop 2000.

[0102] 4.3 Electroporation

[0103] Refer to 1.3 in Example 1 to electrotransform the targeting plasmid pCW633 into LIΔdaldat competent cells. After electroporation and recovery, the bacterial suspension was spread on a BHI plate and cultured at 37°C for 48 - 72 h.

[0104] 4.4 Colony PCR

[0105] Refer to 1.4 in Example 1 to perform colony PCR screening on the single colonies growing on the BHI plate. The screening primers are asd-SX-F / R (the results of colony PCR verification are shown in Figure 3 In d). The asd-SX-F sequence is as shown in SEQ ID NO.22, and the asd-SX-R sequence is as shown in SEQ ID NO.23.

[0106] 4.5 Strain preservation

[0107] The recombinant strain with correct colony PCR verification was promptly preserved and named LIΔdaldat::pCW633.

[0108] Example 5. Determination of the growth curve of LIΔdaldat::pCW633

[0109] Pick the recombinant strain LIΔdaldat::pCW633 preserved at -20°C, streak it on a BHI plate, and culture it 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 LI and LIΔdaldat::pCW633 are as Figure 4 shown. The growth trends of LIΔdaldat::pCW633 and LI are basically the same.

[0110] Example 6. Extract, purify MVs and perform characterization analysis

[0111] 6.1 Large-scale preparation of culture supernatant

[0112] 6.1.1 Resuscitation of bacterial strain

[0113] Pick the recombinant strain LIΔdaldat::pCW633 preserved at -20°C, streak it on a 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 in an incubator at 37°C for 24 h.

[0114] 6.1.2 Large-scale preparation of culture supernatant

[0115] Pick a single colony on the above BHI plate and inoculate it into 5 mL of BHI broth, and incubate it at 37°C with shaking at 200 rpm for 16. 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 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, and 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.

[0116] 6.2 Extraction and purification of MVs

[0117] 6.2.1 Ultrafiltration concentration - ultracentrifugation

[0118] The culture supernatant was filtered through a 0.22-μm filter membrane, and the filtrate was ultrafiltered and concentrated to 35 mL through a 500-KDa cut-off module. The concentrated solution was ultracentrifuged at 140,000 g for 6 h at 4 °C, and the supernatant was discarded. The precipitate was resuspended in 500 μL of 0.01 mol / L PBS (pH 7.2) solution to obtain the crude MVs product.

[0119] 6.2.2 Purification by density gradient centrifugation

[0120] In a 4-mL centrifuge tube, iodixanol solutions with mass concentrations of 45%, 35%, 30%, 25%, 20%, 15%, and 10% were successively layered (from bottom to top), and the crude MVs product was layered on the top. Ultracentrifugation was performed at 180,000 g for 4 h at 4 °C (with the minimum acceleration for speed increase and decrease). After centrifugation, the samples were collected in layers. 20 μL of each layer of the sample was taken, an appropriate amount of SDS-PAGE protein loading buffer was added, and the protein was denatured by boiling for 10 min. The effect of density gradient centrifugation was observed by SDS-PAGE gel electrophoresis (5% stacking gel, 10% separating gel) and Coomassie brilliant blue staining.

[0121] 6.2.3 Washing and desalting

[0122] According to the results of Coomassie brilliant blue staining, the flagellum-free liquid layers were combined, and the volume was made up with 0.01 mol / L PBS (pH 7.2) solution. Ultracentrifugation was performed again at 140,000 g for 6 h at 4 °C, and the supernatant was discarded. The precipitate was resuspended in 50 μL of 0.01 mol / L PBS (pH 7.2) solution to obtain the purified MVs product.

[0123] 6.3 Characterization of MVs

[0124] 6.3.1 Determination of protein concentration by BCA method

[0125] According to the instructions of the BCA protein assay kit, the protein concentration of the purified MVs product was determined to characterize the yield of MVs. The results are as Figure 5 shown. The MVs yield of LIΔdaldat::pCW633 was 441.58 μg / L of culture supernatant, which was 1.44 times that of LI (307.59 μg / L of culture supernatant).

[0126] 6.3.2 TEM characterization of MVs morphological characteristics

[0127] The MVs suspension was dropped onto a 400-mesh copper grid and stained by the 2% phosphotungstic acid negative staining method. Observation was carried out under a transmission electron microscope (80 KV) to characterize the morphological characteristics of MVs. The MVs secreted by the wild strain LI are shown in Figure 6 a, and the MVs secreted by LIΔdaldat::pCW633 are shown in Figure 6 b. As Figure 6As shown, MVs exhibit a typical spherical structure under TEM, with a clean background. Moreover, the MVs secreted by LIΔdaldat::pCW633 have no obvious differences in shape, structure, size, etc. from those of LI. 6.3.3 Dynamic light scattering analysis to characterize the particle size characteristics of MVs

[0128] 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 as the average particle size (Z-average), and show the particle size distribution characteristics of MVs with Intensity PSD. The structure of the dynamic light scattering analysis is shown in Figure 7 , the average particle size of the MVs secreted by LI is 109.2 nm, and the average particle size of the MVs secreted by LIΔdaldat::pCW633 is 115.0 nm, and the particle size distributions of the two are basically the same.

[0129] 6.3.4 SDS-PAGE to characterize the protein composition of MVs

[0130] 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 the sample, electrophorese 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 with Coomassie Brilliant Blue staining solution at room temperature for 1 h and then decolorize until the background is clean, and image through a gel imaging system. As Figure 8 can be seen, the protein composition of the MVs secreted by LIΔdaldat::pCW633 is the same as that of the MVs secreted by LI.

[0131] 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 purpose 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 in sheep Listeria ivanovii ​ It is characterized in that The specific steps are as follows: (1) Knock out the dal , dat genes of the wild strain LI of Listeria monocytogenes, and obtain the strain LIΔ daldat , and prepare the competent cells of LIΔ daldat ; (2) Electroporate the targeting plasmid pCW633 into LIΔ daldat competent cells to obtain the strain LIΔ daldat ::pCW633; (3) Culture LIΔ daldat ::pCW633, centrifuge to obtain the supernatant, and extract and purify to obtain Listeria monocytogenes vesicle LIMVs; The preparation method of the targeting plasmid pCW633 is as follows: Culture Escherichia coli carrying the plasmid pCW630, extract the plasmid pCW630, the nucleotide sequence of which is shown in SEQ ID NO.12, and amplify the vector fragment from the plasmid pCW630 using the primer Vector633-F shown in SEQ ID NO.13 and the 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 the plasmid pCW630 using 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 phly-LM dal , the nucleotide sequence of which is shown in SEQ ID NO.18, and ligate the vector fragment Vector633 with the insert fragment phly-LM dal to obtain the ligation product; Transformation of the (2-B) ligation product into Escherichia coli DH5αΔ asd competent cells, 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. According to the method described in claim 1, It is characterized in that The specific method of step (1) is as follows: (1-1) The wild strain LI of Listeria monocytogenes was used to prepare competent LI, and the targeting plasmid pCW619-LI dal was electrotransformed into competent LI cells. After electrotransformation and recovery, the cells were cultured. Colony PCR screening was performed using the primer pCW619-LIdal-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 LIdal-F shown in SEQ ID NO.3, the primer LIdal-R shown in SEQ ID NO.4, the primer HomoLIdal-F shown in SEQ ID NO.5, and the primer HomoLIdal-R shown in SEQ ID NO.6 to obtain the strain LIΔ dal , and competent cells of LIΔ dal were prepared; (1-2) The targeting plasmid pCW619-LI dat was electrotransformed into LIΔ dal competent cells. After electrotransformation recovery and culture, colony PCR screening was performed using the primer pCW619-LIdat-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 performed using the primer LIdat-F shown in SEQ ID NO.8, the primer LIdat-R shown in SEQ ID NO.9, the primer HomoLIdat-F shown in SEQ ID NO.10, and the primer HomoLIdat-R shown in SEQ ID NO.11 to obtain the strain LIΔ daldat , and LIΔ daldat competent cells were prepared.

3. According to the method described in claim 2, It is characterized in that In step (1-1), the preparation method of the target plasmid pCW619-LI dal is as follows: Streak the Escherichia coli strain on an LB solid medium supplemented with 100 μg / mL ampicillin and culture it at 37 °C for 16 hours; pick a single colony and inoculate it into an LB liquid medium supplemented with 100 μg / mL ampicillin, incubate it at 37 °C with shaking at 200 rpm for 16 hours, and extract the plasmid pCW619-LI dal dal .​ 4. According to the method described in claim 1, It is characterized in that In step (2), the preparation method of strain LIΔ daldat ::pCW633 is as follows: The targeting plasmid pCW633 is electrotransformed into LIΔ daldat competent cells. After electrotransformation and recovery, 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 LIΔ daldat ::pCW633.

5. According to the method described in claim 1, It is characterized in that In step (3), LIΔ daldat The specific method for culturing ::pCW633 is as follows: Streak the strain LIΔ daldat ::pCW633 onto a BHI plate and culture at 37°C for 24 hours; Pick a single colony and inoculate it into 5 mL of BHI broth, and incubate at 37°C with shaking at 200 rpm for 16 hours; Pipette 250 μL of the bacterial solution and inoculate it into 25 mL of BHI broth, and incubate 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 at 37°C with shaking at 200 rpm for 16 hours.

6. According to the method described in claim 1, It is characterized in that In step (3), the process conditions for centrifugation are: centrifuge at 4°C and 12,000 g for 10 minutes.

7. According to the method described in claim 1, It is characterized in that 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) Subject the crude extract to ultra-high speed centrifugation using multi-layer iodixanol solutions with different concentrations, collect the layers separately, combine the flagella-free liquid layers, make up the volume with 0.01 mol / L PBS, perform ultra-high speed centrifugation again to obtain the precipitate, and resuspend it with 0.01 mol / L PBS to obtain the product.

8. According to the method described in claim 7, It is characterized in that 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: centrifuge at 4°C and 140,000 g for 6 hours; In step (3-b), the mass concentrations of the iodixanol solutions are 45%, 35%, 30%, 25%, 20%, 15%, and 10% successively from bottom to top and are laid into the centrifuge tube, and the crude extract is laid on the top layer; In step (3-b), the process conditions for ultra-high speed centrifugation are: centrifuge at 4°C and 180,000 g for 4 hours; the process conditions for the second ultra-high speed centrifugation are: centrifuge at 4°C and 140,000 g for 6 hours.

9. Application of the method described in any one of claims 1 to 8 in the preparation of a preparation using LI MVs as a carrier.

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