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

Abstract

The present invention relates to a method for improving the yield of attenuated Listeria monocytogenes membrane vesicles and its application. Starting from the attenuated Listeria monocytogenes LMΔactAplcB, an attenuated strain LMΔactAplcBdaldat with dal and dat genes knocked out is constructed, and a complementing plasmid pCW633 carrying the dal gene is constructed, and the plasmid is electrotransferred into the attenuated nutrient-deficient strain LMΔactAplcBdaldat to construct a stable recombinant strain LMΔactAplcBdaldat::pCW633. Compared with the method of antibiotic treatment, the yield of secreted MVs of the recombinant strain LMΔactAplcBdaldat::pCW633 constructed by the present invention is more significantly increased. The present invention can solve the problem of low yield of attenuated LM MVs and can be used for basic and applied research related to attenuated LM MVs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for increasing the yield of attenuated Listeria monocytogenes membrane vesicles and an application thereof. Background Art

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

[0003] At present, the research on BMVs is mostly focused on OMVs secreted by Gram-negative bacteria, including Escherichia coli, Neisseria meningitidis, Pseudomonas aeruginosa, etc. However, it is worth noting that OMVs contain Gram-negative bacteria. - The presence of lipopolysaccharide (LPS), a pyrogen that is a unique component of bacterial cell membranes, makes the safety of OMVs-based biological preparations questionable, which also limits the application of OMVs to a certain extent. + MVs produced by G. + Biological agents developed based on bacterial MVs have more advantages than OMVs. However, there are relatively few studies based on MVs, and only a few G. +The main limiting factor is G + The production 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 genus of G + Non-spore-forming short bacteria, intracellular parasites, have been found in many species, including Listeria monocytogenes (LM) and Listeria ivanovii (LI). LM and LI can produce a variety of virulence factors such as internalization and hemolysin, promote phagocytosis and escape from lysosomes, and stimulate cellular and humoral immune responses. In 2013, Lee et al. first discovered that LM can also produce MVs. Studies have found that LM MVs are similar to other MVs, with good biosafety, high immunogenicity, and the ability to load exogenous antigens, but they also have the problem of low yield. Summary of the invention

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

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

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

[0008] (1) knocking out the dal and dat genes of the attenuated Listeria monocytogenes strain LMΔactAplcB to obtain the strain LMΔactAplcBdaldat, and preparing LMΔactAplcBdaldat competent cells;

[0009] (2) The targeting plasmid pCW633 was electroporated into LMΔactAplcBdaldat competent cells to obtain the strain LMΔactAplcBdaldat::pCW633;

[0010] (3) Cultivating LMΔactAplcBdaldat::pCW633, taking the supernatant by centrifugation, and extracting and purifying the attenuated Listeria monocytogenes membrane vesicles, i.e., attenuated LM MVs.

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

[0012] (1-1) The attenuated Listeria monocytogenes strain LMΔactAplcB was used to prepare competent cells LMΔactAplcB, and the targeting plasmid pCW619-LMdal was electroporated into the LMΔactAplcB competent cells. After electroporation and resuscitation, the cells were cultured, and colony PCR screening was performed using the primers pCW619-Lmdal-F shown in SEQ ID NO.1 and the primers pCW619-R shown in SEQ ID NO.2. After homologous recombination, colony PCR screening was performed using the primers LMdal-F shown in SEQ ID NO.3, the primers LMdal-R shown in SEQ ID NO.4, the primers HomoLMdal-F shown in SEQ ID NO.5, and the primers HomoLMdal-R shown in SEQ ID NO.6 to obtain the strain LMΔactAplcBdal, and LMΔactAplcBdal competent cells were prepared;

[0013] (1-2) The targeting plasmid pCW619-LMdat was electroporated into LMΔactAplcBdal competent cells, and the cells were cultured after electroporation and resuscitation. Colony PCR screening was performed using primers pCW619-Lmdat-F as shown in SEQ ID NO.7 and primers pCW619-R as shown in SEQ ID NO.2. Homologous recombination was performed using primers LMdat-F as shown in SEQ ID NO.8, primers LMdat-R as shown in SEQ ID NO.9, primers HomoLMdat-F as shown in SEQ ID NO.10, and primers HomoLMdat-R as shown in SEQ ID NO.11 to obtain strain LMΔactAplcBdaldat, and LMΔactAplcBdaldat competent cells were prepared.

[0014] As one of the further preferred technical schemes, 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 is streaked and inoculated into an LB solid culture medium supplemented with 100 μg / mL ampicillin, and cultured at 37°C for 16 hours; a single colony is picked and inoculated into an LB liquid culture medium supplemented with 100 μg / mL ampicillin, and incubated at 37°C, 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 LMΔactAplcBdaldat::pCW633 is as follows: the targeting plasmid pCW633 is electroporated into LMΔactAplcBdaldat competent cells, and the cells are cultured after electroporation and recovery, and colony PCR screening is performed using primers asd-SX-F as shown in SEQ ID NO.22 and primers asd-SX-R as shown in SEQ ID NO.23 to obtain the strain LMΔactAplcBdaldat::pCW633.

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

[0017] (2-A) Cultivating Escherichia coli carrying plasmid pCW630, extracting plasmid pCW630, whose nucleotide sequence is shown in SEQ ID NO.12, amplifying vector fragment Vector633 from plasmid pCW630 using primers Vector633-F shown in SEQ ID NO.13 and primers Vector633-R shown in SEQ ID NO.14, whose nucleotide sequence is shown in SEQ ID NO.17; amplifying insert fragment phly-LM dal from plasmid pCW630 using primers phly-LM dal-F shown in SEQ ID NO.15 and primers phly-LM dal-R shown in SEQ ID NO.16, whose nucleotide sequence is shown in SEQ ID NO.18, and ligating vector fragment Vector633 and insert fragment phly-LM dal to obtain a ligation product;

[0018] (2-B) The ligation product was transformed into Escherichia coli DH5αΔasd competent cells, and colony PCR screening was performed using primers pCW633-SX-F shown in SEQ ID NO.19 and primers pCW633-SX-R shown in SEQ ID NO.20 to extract 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 LMΔactAplcBdaldat::pCW633 is as follows: streak the strain LMΔactAplcBdaldat::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 shake it at 37°C, 200 rpm for 16 hours; draw 250 μL of fresh bacterial solution and inoculate it into 25 mL of BHI broth, and shake it at 37°C, 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 shake it at 37°C, 200 rpm for 16 hours.

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

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

[0022] (3-a) The supernatant was filtered through a 0.22 μm filter membrane, concentrated by ultrafiltration, and the precipitate was collected by ultrahigh-speed centrifugation. The precipitate was resuspended in 0.01 mol / L PBS (pH 7.2) to obtain a crude product;

[0023] (3-b) The crude product was subjected to ultra-high speed centrifugation using multiple layers of iodixanol solutions of different concentrations, and the layers were collected and the liquid layers without flagella were combined. The volume was supplemented with 0.01 mol / L PBS (pH 7.2), and the precipitate was collected by ultra-high speed centrifugation again, and resuspended in 0.01 mol / L PBS (pH 7.2).

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

[0025] As one of the further preferred technical solutions, in step (3-b), the mass concentration of the iodixanol solution is 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.

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

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

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

[0029] In order to solve the problem of low yield of attenuated Listeria monocytogenes MVs, the present invention increases the yield of attenuated LM MVs from the perspective of reducing the cross-linking degree of the peptidoglycan layer of the bacterial cell wall. In the process of bacterial cell wall synthesis, D-alanine (D-alanine, D-Ala) plays a vital role in the cross-linking between the peptidoglycan layers. The synthesis of D-Ala is related to the dal and dat genes. The dal gene controls the synthesis of alanine racemase (Alanine racemase, Alr), which can convert L-alanine into D-Ala, while the dat gene controls the synthesis of D-alanine transaminase (D-amino acid aminotransferase, D-AAT), which can convert D-glutamate and pyruvate into D-Ala and α-ketoglutarate through transamination. When the dal and dat genes on the bacterial genome are knocked out, the bacteria cannot form cell walls and cannot grow in a culture medium without exogenous D-Ala addition. Therefore, the present invention adds the dal gene to the plasmid and back-fills it into the dal and dat deletion strains, thereby reducing the cross-linking degree of the peptidoglycan layer in the bacterial cell wall as much as possible while maintaining bacterial growth, thereby achieving the purpose of increasing the yield of MVs. Currently, there is no method for increasing the yield of MVs related to the dal and dat genes, so the present invention is original.

[0030] The present invention starts from the attenuated Listeria LMΔactAplcB, constructs the LMΔactAplcBdaldat attenuated strain with dal and dat genes knocked out, and constructs the complementing plasmid pCW633 carrying the dal gene, and electrotransfers it into the attenuated nutritional deficiency strain LMΔactAplcBdaldat to construct a stable recombinant strain LMΔactAplcBdaldat::pCW633. The MVs of the recombinant strain are collected by the LM MVs extraction (ultrafiltration concentration-ultracentrifugation method) and purification (density gradient centrifugation method) scheme established and optimized by us. Then, the total protein concentration of the recombinant MVs is determined by a BCA kit, 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-type LM, the yield of MVs secreted by the recombinant strain LMΔactAplcBdaldat::pCW633 can be increased by 4.22 times, and the shape structure, particle size and protein components of the MVs secreted by the recombinant strain are not significantly different from those of the MVs 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 untreated group, respectively. Therefore, the yield of MVs secreted by the recombinant strain LMΔactAplcBdaldat::pCW633 constructed by the present invention is more significantly increased. The present invention can solve the problem of low yield of attenuated LM MVs, and can be used for related basic research and applied research based on attenuated LM MVs.

[0031] The present invention can be used for basic research related to attenuated LM MVs, including multi-omics research on attenuated LM MVs (proteomics, lipidomics, metabolomics, etc.), research on the potential biological functions of attenuated LM MVs (pathogenicity, signal transduction, quorum sensing, pressure stress, etc.), and related preparation applications using attenuated LM MVs as carriers (drug delivery, antigen targeting, regulation of immune response, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0033] Figure 1Schematic diagram of the plasmids involved in preparing the recombinant strain LMΔactAplcBdaldat::pCW633 of the present invention, wherein 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 Schematic diagram of the construction process of the recombinant strain LMΔactAplcBdaldat::pCW633 of the present invention;

[0035] Figure 3 The colony PCR verification results of the recombinant strain LMΔactAplcBdaldat::pCW633 prepared by the present invention, wherein M is a 250bp DNA Marker; in the figure a, 1 and 2 are respectively amplified by primers HomoLMdal-F / R for LM and LMΔactAplcBdaldat::pCW633; in the figure b, 1 and 2 are respectively amplified by primers HomoLMdat-F / R for LM and LMΔactAplcBdaldat::pCW633; in the figure c, 1 and 2 are respectively amplified by primers pCW633-SX-F / R for plasmid pCW630 and Escherichia coli pCW633; in the figure d, 1 and 2 are respectively amplified by primers asd-SX-F / R for plasmid pCW633 and LMΔactAplcBdaldat::pCW633;

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

[0037] Figure 5 Comparison of the yield of MVs secreted by the wild strain LM and the recombinant strain LMΔactAplcBdaldat::pCW633 in the present invention, ** indicates <0.05;

[0038] Figure 6 These are transmission electron microscopic images of MVs secreted by the wild strain LM and the recombinant strain LMΔactAplcBdaldat::pCW633 in the present invention, used to observe the morphology of MVs; Figure a shows MVs secreted by the wild strain LM; Figure b shows MVs secreted by the recombinant strain LMΔactAplcBdaldat::pCW633;

[0039] Figure 7 Dynamic light scattering analysis of MVs secreted by the wild-type LM and the recombinant strain LMΔactAplcBdaldat::pCW633 of the present invention is used to characterize the particle size distribution of MVs;

[0040] Figure 8 This is the SDS-PAGE electrophoresis diagram of MVs secreted by the wild strain LM and the recombinant strain LMΔactAplcBdaldat::pCW633 in the present invention, which is used to analyze the protein components of MVs, M is a protein molecular weight marker; 1 is the LM wild strain; 2 is the recombinant strain LMΔactAplcBdaldat::pCW633.

[0041] Fig. 9 Comparison of crude extract yields of MVs secreted by the wild strain LM under different antibiotic treatment conditions. DETAILED DESCRIPTION

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

[0043] Example 1. Construction of LMΔactAplcBdal strain

[0044] 1.1 Preparation of competent cells of attenuated strain LMΔactAplcB

[0045] 1.1.1 Bacterial strain recovery

[0046] The attenuated strain LMΔactAplcB (Lei Y, Zhou Y, Zhang Y, Liu S, Tian S, OuQ, 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 was streaked onto a Brain-Heart Infusion Broth (BHI) plate and inverted in a 37°C incubator for 24 hours. A single colony was picked and inoculated onto another BHI plate, which was inverted in a 37°C incubator for 24 hours.

[0047] 1.1.2 Preparation of competent cells

[0048] Pick 3-4 single colonies on the above plate and inoculate them into 15mL BHI broth (containing 0.5mol / L sucrose), and shake at 37℃200rpm for 16 hours. The next morning, inoculate the above 15mL fresh bacterial solution into 250mL BHI broth (containing 0.5mol / L sucrose), and shake at 37℃200rpm. Adjust to zero with BHI broth (containing 0.5mol / L sucrose), and monitor the OD600 value of the bacterial solution regularly. When the OD600 value reaches 0.4, add penicillin G solution (final concentration is 12.5μg / mL) and continue to shake. When the OD600 value of the bacterial solution reaches 0.7 or begins to decrease, collect the bacteria. Divide the bacterial solution into 50mL centrifuge tubes, centrifuge at 4℃13000rpm for 5min, and discard the supernatant. Add 20mL of pre-cooled 0.5M sucrose solution to resuspend the bacterial pellet, centrifuge at 4℃10000rpm for 10min, discard the supernatant, and repeat this step once. Resuspend the bacterial pellet with 300μL of 0.5mol / L sucrose, divide into 50μL / tube and store at -80℃. Collect the bacterial cells on ice throughout the process.

[0049] 1.2 Preparation of targeting plasmid pCW619-LMdal (NCBI accession no.MN528127)

[0050] 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. Steria 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 was streaked and inoculated into LB (Luria-Bertani) solid medium (hereinafter referred to as LA plate) supplemented with 100 μg / mL ampicillin (Ampicillin, Amp) and cultured in an incubator at 37°C for 16 hours. A single colony was inoculated into 5 mL of LB liquid medium (hereinafter referred to as LA broth) supplemented with 100 μg / mL Amp and incubated at 37°C at 200 rpm for 16 hours. According to the instructions of the Omega plasmid extraction kit, the plasmid pCW619-LMdal was extracted and eluted with an appropriate amount of sterile ddH2O. The plasmid concentration and purity were determined using Nanodrop 2000. Figure 1 Middle a.

[0051] 1.3 Electroporation

[0052] Precool 10μL and 200μL pipette tips and electroporation cups at -20℃, and preheat 1000μL pipette tips and BHI broth (containing 1mol / L sucrose) at 37℃. Thaw LMΔactAplcB competent cells and targeting plasmid pCW619-LMdal on ice. Pipette 5μL plasmid solution dropwise and add to the corresponding 50μL competent cells, mix gently with fingertips, and ice bath for 5min. Transfer the above mixture to the electroporation cup and ice bath for 5min. Electroporate on the electroporator at 1500V for 5ms, remove the electroporation cup, and ice bath for 5min. Add 700μL BHI broth (containing 1mol / L sucrose) to the electroporation cup, mix the bacteria and then aspirate into a 1.5mL EP tube. Resuscitate at 37℃ and 200rpm for 2h. After the recovery, centrifuge at 12000rpm for 2min, discard about 600μL supernatant, and resuspend the bacterial pellet. The resuspended bacterial solution was spread onto BHI solid culture medium (hereinafter referred to as BE3 plate) supplemented with 3 μg / mL erythromycin (Ery), and cultured in an incubator at 37°C for 48h-72h.

[0053] 1.4 Screening after electroporation

[0054] Pick a single colony on the BE3 plate and add it to 20 μL 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 pCW619-Lmdal-F sequence is as shown in SEQ ID NO.1, and the pCW619-R sequence is as shown in SEQ ID NO.2. Amplify according to conventional PCR conditions. After amplification, analyze by agarose gel electrophoresis (mass concentration 1% agarose gel, 90V voltage).

[0055] 1.5 Homologous recombination

[0056] The single colony that was successfully electroporated was streaked onto a D-BE3 plate, which was a BHI solid medium supplemented with 3 μg / mL erythromycin (Ery) and 200 μg / mL D-alanine (hereinafter referred to as D-BE3 plate), and cultured in a 42°C incubator for three consecutive passages. The bacterial moss 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, and passaged six times. The sixth generation bacterial solution was gradiently diluted to 10 with 0.01 mol / L PBS (pH 7.2) solution. -6 , 100 μL of the dilution was spread on BHI solid medium supplemented with 200 μg / mL D-alanine (hereinafter referred to as D-BHI plate) and cultured in a 37°C incubator for 48 h.

[0057] 1.6 Screening

[0058] Pick a single colony on the above D-BHI plate and streak it onto a D-BE3 plate and a D-BHI plate at the same time and culture at 37°C for 24 hours. Select a single colony that does not grow on the D-BE3 plate but grows on the D-BHI plate for colony PCR screening. Pick a small amount of bacterial moss on the D-BHI plate and add it to 20μL 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 colony PCR verification results for details). Figure 3 a), amplify under conventional PCR conditions. After amplification, analyze by agarose gel electrophoresis (mass concentration 1% agarose gel, 90V voltage). LMdal-F sequence is SEQ ID NO.3, LMdal-R sequence is SEQ ID NO.4, HomoLMdal-F sequence is SEQ ID NO.5, and HomoLMdal-R sequence is SEQ ID NO.6.

[0059] 1.7 Sequencing

[0060] The amplification product of HomoLMdal-F / R primers was sent to the company for sequencing. The correctly sequenced strain was named LMΔactAplcBdal and the strain was preserved in time.

[0061] Example 2. Construction of LMΔactAplcBdaldat strain

[0062] 2.1 Preparation of LMΔactAplcBdal competent cells

[0063] Prepare LMΔactAplcBdal competent cells with reference to 1.1 in Example 1. 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 targeting plasmid pCW619-LMdat (NCBI accession no. MN 528128)

[0065] Referring to 1.2 in Example 1, prepare the targeting plasmid pCW619-LMdat (Lei Y, Zhou Y, Zhang Y, Liu S, Tian S, Ou Q, Liu T, Huang H, Tang T, Wang CA 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 Middle b.

[0066] 2.3 Electroporation

[0067] The targeting plasmid pCW619-LMdat was electroporated into LMΔactAplcBdal competent cells according to 1.3 of Example 1. The bacterial suspension after electroporation was spread on D-BE3 plates and cultured at 37°C for 48-72h.

[0068] 2.4 Screening after electroporation

[0069] The single colonies grown on the D-BE3 plate were screened by colony PCR with reference to 1.4 of Example 1. The screening primers were pCW619-Lmdat-F / pCW619-R, pCW619-Lmdat-F was as shown in SEQ ID NO.7, and the pCW619-R sequence was as shown in SEQ ID NO.2.

[0070] 2.5 Homologous recombination

[0071] Homologous recombination was performed according to step 1.5 of Example 1.

[0072] 2.6 Screening

[0073] Pick the single colonies on the D-BHI plate and streak them on the BHI plate, D-BE3 plate and D-BHI plate at the same time, and culture them in a 37°C incubator for 24 hours. Select the single colonies that do not grow on the BHI plate and D-BE3 plate but grow on the D-BHI plate for colony PCR screening.

[0074] Pick a small amount of bacterial moss from the D-BHI plate and add it to 20 μL 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 (the colony PCR verification results are shown in Figure 3b). The LMdat-F sequence is SEQ ID NO.8, the LMdat-R sequence is SEQ ID NO.9, the HomoLMdat-F sequence is SEQ ID NO.10, and the HomoLMdat-R sequence is SEQ ID NO.11.

[0075] 2.7 Sequencing

[0076] The amplification product of HomoLMdat-F / R primers was sent to the company for sequencing. The correctly sequenced strain was named LMΔactAplcBdaldat and the strain was preserved in time.

[0077] Example 3. Construction of complementing plasmid pCW633

[0078] 3.1 Preparation of competent E. coli DH5αΔasd (CaCl2 method)

[0079] 3.1.1 Bacterial recovery

[0080] The Escherichia coli DH5αΔasd strain stored at -20°C was streaked onto 5 mL of LB solid medium supplemented with 50 μg / mL diaminopimelic acid (DAP) (hereinafter referred to as DAP-LB plate) and cultured at 37°C for 16 hours.

[0081] 3.1.2 Preparation of competent E. coli DH5αΔasd

[0082] Pick a single colony and inoculate it into 5mL LB liquid culture medium (hereinafter referred to as DAP-LB broth) supplemented with 50μg / mL DAP, and shake at 37℃200rpm for 16 hours. Pipette 1mL of fresh bacterial solution and inoculate it into 50mL DAP-LB broth, shake at 37℃200rpm until the OD600 value is about 0.5 to collect the bacteria. Transfer the bacterial solution to a 50mL centrifuge tube, ice bath for 30min, centrifuge at 4℃8000rpm for 3min, and discard the supernatant. Add 10mL pre-cooled 0.1mol / L CaCl2 to resuspend, centrifuge at 4℃6000rpm for 3min, and discard the supernatant. Resuspend with 1mL pre-cooled 0.1mol / L CaCl2, divide into 50μL / tube and store at -80℃.

[0083] 3.2 Preparation of vector fragment and insert fragment

[0084] 3.2.1 Extraction of plasmid pCW630

[0085] Pick out the E. coli strain carrying the pCW630 plasmid stored at -20℃ and streak it onto an LB plate and incubate it at 37℃ for 16 hours to revive the strain. Pick out a single colony and inoculate it into 5mL LB broth and shake it at 37℃ and 200rpm for 16 hours. Follow the instructions of the Omega plasmid extraction kit to extract the plasmid pCW630 and elute it with 35μL sterile ddH2O. Use Nanodrop2000 to determine the plasmid concentration and purity. The sequence of plasmid pCW630 is shown in SEQ ID NO.12, and the schematic diagram of the plasmid is shown in Figure 1 Middle c.

[0086] 3.2.2 Preparation of vector fragment and insert fragment

[0087] Primers Vector633-F / Vector633-R are used to amplify vector fragment Vector633 from plasmid pCW630, and primers phly-LM dal-F / R are used to amplify insert fragment phly-LM dal from plasmid pCW630, and the amplification can be performed according to conventional PCR conditions. After the amplification, each fragment is separated by agarose gel electrophoresis (mass concentration 1.5% agarose gel, 90V voltage), and the fragment is purified and recovered by gel cutting using Omega EZNAGel Extraction Kit. The sequence of primer Vector633-F is such as SEQ ID NO.13, the sequence of Vector633-R is such as SEQ ID NO.14, the sequence of phly-LM dal-F is such as SEQ ID NO.15, the sequence of phly-LM dal-R is such as SEQ ID NO.16, the sequence of vector fragment Vector633 is such as SEQ ID NO.17, and the sequence of insert fragment phly-LM dal is such as SEQ ID NO.18.

[0088] 3.3 Connection conversion

[0089] 3.3.1 Connection

[0090] Use ABclonal MultiF Seamless Assembly Mix (RK21020) seamless ligation kit to connect the vector fragment Vector633 and the insert fragment phly-LM dal. For specific operations, see the instructions.

[0091] 3.3.2 Conversion

[0092] Thaw E. coli DH5αΔasd competent cells on ice, add 5μL of ligation product, mix gently with fingertips, and let stand on ice for 30min. Heat shock in 42℃ water bath for 45s, quickly transfer to ice, and let stand for 2min. Add 700μL LB broth and resuscitate at 37℃ 200rpm for 1h. After resuscitation, centrifuge at 12000rpm for 2min, discard about 600μL supernatant, and resuspend the bacterial pellet. According to experimental needs, aspirate different volumes of resuspended bacterial solution and apply it to LB plate and culture at 37℃ for 16 hours.

[0093] 3.4 Colony PCR

[0094] Pick a single colony grown on the LB plate and add it to 20 μL ddH2O to prepare a bacterial suspension. Use this bacterial suspension as a template for colony PCR screening. Primer pCW633-SX-F / R is used for colony PCR screening (see the colony PCR verification results for details). Figure 3 (c) pCW633-SX-F sequence is shown in SEQ ID NO.19, and pCW633-SX-R sequence is shown in SEQ ID NO.20.

[0095] 3.5 Sequencing

[0096] The correct single colony verified by colony PCR was selected and inoculated into LB broth, and the plasmid was extracted and sent to a sequencing company for sequencing. The correct plasmid was named pCW633 and stored in E. coli pCW633. The sequence of plasmid pCW633 is shown in SEQ ID NO.21, and the schematic diagram of the plasmid is shown in Figure 1 Middle d.

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

[0098] 4.1 Preparation of LMΔactAplcBdaldat competent cells

[0099] Prepare LMΔactAplcBdaldat competent cells with reference to 1.1 in Example 1. Note that the culture medium used in this step is D-BHI plate and D-BHI broth (containing 0.5 mol / L sucrose).

[0100] 4.2 Preparation of complementation plasmid pCW633

[0101] Pick the E. coli strain pCW633 stored at -20℃ and streak it onto LB plate and incubate it at 37℃ for 16 hours to revive the strain. Pick a single colony and inoculate it into 5mL LB broth and shake it at 37℃ and 200rpm for 16 hours. According to the instructions of Omega plasmid extraction kit, extract plasmid pCW633 and elute it with 35μL sterile ddH2O. Use Nanodrop 2000 to determine the concentration and purity of the plasmid.

[0102] 4.3 Electroporation

[0103] The complementing plasmid pCW633 was electroporated into LMΔactAplcBdaldat competent cells according to 1.3 of Example 1. The bacterial suspension after electroporation was spread on BHI plates and cultured at 37°C for 48-72 hours.

[0104] 4.4 Colony PCR

[0105] Refer to 1.4 in Example 1 for colony PCR screening of single colonies grown on BHI plates. The screening primers are asd-SX-F / R (the colony PCR verification results are shown in Figure 3 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 Preservation of strains

[0107] The complemented strain verified correctly by colony PCR was promptly preserved and named LMΔactAplcBdaldat::pCW633.

[0108] Example 5. LMΔactAplcBdaldat::pCW633 growth curve assay

[0109] Pick the recombinant strain LMΔactAplcBdaldat::pCW633 stored at -20℃ and streak it onto a BHI plate, then invert it and culture it in a 37℃ incubator for 24h. Pick a single colony and inoculate it into 5mL BHI broth, and shake it at 37℃ and 200rpm for 16 hours. Take an appropriate amount of bacterial liquid and inoculate it into 50mL BHI broth, adjust the absorbance value OD600 at 600nm to 0.06, shake it at 37℃ and 200rpm, measure OD600 every 1h, and draw a growth curve based on OD600. LM and

[0110] The growth curve of LMΔactAplcBdaldat::pCW633 is shown in Figure 4 As shown in Figure 2, the growth trend of LMΔactAplcBdaldat::pCW633 is basically consistent with that of LM.

[0111] Example 6. Extraction, purification and characterization of MVs

[0112] 6.1 Preparation of large volume culture supernatant

[0113] 6.1.1 Bacterial strain recovery

[0114] Pick the recombinant strain LMΔactAplcBdaldat::pCW633 stored at -20℃ and streak it onto a BHI plate, then invert it in a 37℃ incubator and culture it for 24h. Pick a single colony and inoculate it onto a new BHI plate, then invert it in a 37℃ incubator and continue to culture it for 24h. 6.1.2 Prepare the culture supernatant in large volume

[0115] Pick a single colony from the BHI plate and inoculate it into 5 mL BHI broth, shake at 37°C 200 rpm for 16 hours. Take 250 μL of fresh bacterial solution and inoculate it into 25 mL BHI broth, shake at 37°C 200 rpm for 16 hours. After adjusting the OD600 value of the bacterial solution to about 1.0, take 10 mL of the bacterial solution and inoculate it into 1 L BHI broth, shake at 37°C 200 rpm for 16 hours. Centrifuge at 4°C 12000g for 10 minutes and collect the culture supernatant.

[0116] 6.2MVs extraction and purification

[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 cutoff module. The concentrate was ultracentrifuged at 140,000 g for 6 h at 4 °C, and the supernatant was discarded. The precipitate was resuspended in 500 μL 0.01 mol / L PBS (pH 7.2) solution, which was the crude MVs extract.

[0119] 6.2.2 Density gradient centrifugation purification

[0120] In a 4mL centrifuge tube, iodixanol solutions with mass concentrations of 45%, 35%, 30%, 25%, 20%, 15%, and 10% were layered in sequence (from bottom to top), and the crude MVs extract was layered on the top layer. The samples were ultracentrifuged at 180000g at 4°C for 4h (with the smallest acceleration). After the centrifugation, the samples were collected in layers. 20μL of sample was taken from each layer, and an appropriate amount of SDS-PAGE protein loading buffer was added. The protein was denatured by boiling for 10min, and the density gradient centrifugation effect was observed by SDS-PAGE gel electrophoresis (5% concentrated gel, 10% separation gel) and Coomassie brilliant blue staining.

[0121] 6.2.3 Washing and desalting

[0122] According to the results of Coomassie Brilliant Blue staining, the layers without flagella were combined, the volume was supplemented with 0.01 mol / L PBS (pH 7.2) solution, and ultracentrifuged again at 140000g for 6 h at 4°C, and the supernatant was discarded. The precipitate was resuspended with 50 μL 0.01 mol / L PBS (pH 7.2) solution to obtain the purified MVs product.

[0123] 6.3MVs Characterization

[0124] 6.3.1 BCA method for protein concentration measurement

[0125] According to the instructions of the BCA protein assay kit, the protein concentration of the MVs purified product was determined to characterize the yield of MVs. Figure 5 As shown, the MVs yield of LMΔactAplcBdaldat::pCW633 was 203.20 μg / L culture supernatant, which was 4.22 times that of LM MVs (48.17 μg / L culture supernatant).

[0126] 5.3.2TEM characterization of MVs morphology

[0127] The MVs suspension was dripped onto a 400-mesh copper grid and stained using 2% phosphotungstic acid negative staining. The morphological characteristics of the MVs were characterized under a transmission electron microscope (80 KV). Figure 6 In a, MVs secreted by LMΔactAplcBdaldat::pCW633 are shown in Figure 6 b. Figure 6 As shown, MVs showed typical spherical structures under TEM with a clean background, and the shape, structure, and size of MVs secreted by LMΔactAplcBdaldat::pCW633 were not significantly different from those of LM.

[0128] 6.3.3 Dynamic light scattering analysis to characterize MVs particle size characteristics

[0129] The MVs suspension was diluted to 1 mL with 0.01 mol / L PBS (pH 7.2) at a ratio of 1:20, added to a cuvette and measured by a particle size analyzer (Malvern Zetasizer Nano ZS). The average particle size (Z-average) was used to report the average particle size of MVs, and the intensity PSD was used to show the particle size distribution characteristics of MVs. Dynamic light scattering analysis structure see Figure 7 The average particle size of MVs secreted by LM(WT) was 99.4 nm, and the average particle size of LMΔactAplcBdaldat::pCW633MVs was 105.4 nm, and the particle size distribution of the two was basically the same.

[0130] 6.3.4 Characterization of MVs protein composition by SDS-PAGE

[0131] Take 10 μL MVs sample, add appropriate amount of SDS-PAGE protein loading buffer, boil for 10 minutes to denature the protein. Prepare SDS-PAGE gel (5% concentrated gel, 10% separation gel), load the sample, run electrophoresis at 80V for 30 minutes, then switch to 120V for 1 hour. After electrophoresis, stain with Coomassie Brilliant Blue staining solution at room temperature for 1 hour, then decolorize until the background is clear, and image with a gel imaging system. Figure 8 It can be seen that the protein composition of MVs secreted by LMΔactAplcBdaldat::pCW633 is consistent with that of MVs secreted by LM.

[0132] Example 7. Secretion of MVs under different antibiotic treatment conditions

[0133] 7.1 Preparation of large volume culture supernatant

[0134] 7.1.1 Bacterial strain recovery

[0135] Pick out the strain LM stored at -20℃ and streak it onto a BHI plate and culture it at 37℃ for 24h. Pick out a single colony and inoculate it onto another BHI plate and culture it in a 37℃ incubator for 24h.

[0136] 7.1.2 Preparation of large volume culture supernatant

[0137] Pick a single colony from the BHI plate and inoculate it into 5mL BHI broth, shake at 37℃200rpm for 16 hours. Take 250μL of fresh bacterial solution and inoculate it into 25mL BHI broth, shake at 37℃200rpm for 16h. After adjusting the OD600 value of the above bacterial solution to about 1.0, take 10mL of bacterial solution and inoculate it into 1L BHI broth with different antibiotics, shake at 37℃200rpm for 16h. Centrifuge at 4℃12000g for 10min and collect the culture supernatant. Among them, the ampicillin (Amp) group was BHI broth containing 0.1221 μg / mL Amp, the gentamicin (Gen) group was BHI broth containing 0.4883 μg / mL Gen, the erythromycin (Ery) group was BHI broth containing 0.0610 μg / mL Ery, and the trimethoprim (Tmp) group was BHI broth containing 1.953 μg / mL Tmp.

[0138] 7.2 MVs extraction and characterization

[0139] 7.2.1 Ultrafiltration concentration-ultracentrifugation

[0140] 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 cutoff module. The concentrate was ultracentrifuged at 140,000 g for 6 h at 4 °C, and the supernatant was discarded. The precipitate was resuspended in 500 μL 0.01 mol / L PBS (pH 7.2) solution, which was the crude MVs extract.

[0141] 7.2.2 BCA method for protein concentration measurement

[0142] According to the instructions of the BCA protein assay kit, the protein concentration of the crude MVs extract was determined to characterize the yield of MVs. Fig. 9 As shown, compared with the yield of LM MVs, the MVs yield of the Amp treatment group was 1.47 times that of the LM group, the MVs yield of the Ery treatment group was 1.36 times that of the LM group, while the MVs yield of the Gen treatment group and the Tmp treatment group was reduced compared with the LM group, which were 0.47 times and 0.85 times that of the LM group, respectively.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A method for increasing the yield of attenuated Listeria monocytogenes membrane vesicles, characterized in that: The specific steps are as follows: (1) Knockout of Listeria monocytogenes ( Listeria monocytogenes ) attenuated strain LMΔ actAplcB of dal , dat gene, and obtained strain LMΔ actAplcBdaldat , and prepare LMΔ actAplcBdaldat Competent cells; (2) Electroporate the targeting plasmid pCW633 into LMΔ actAplcBdaldat In competent cells, strain LMΔ was obtained actAplcBdaldat ::pCW633; (3) Cultivation of LMΔ actAplcBdaldat ::pCW633, centrifuge and take the supernatant, extract and purify to obtain attenuated Listeria monocytogenes membrane vesicles, i.e. attenuated LM MVs; The preparation method of targeting plasmid pCW633 is as follows: (2-A) Cultivating Escherichia coli carrying the pCW630 plasmid, extracting the plasmid pCW630, whose nucleotide sequence is shown in SEQ ID NO.12, and amplifying the vector fragment from the plasmid pCW630 using the primers Vector633-F shown in SEQ ID NO.13 and the primers Vector633-R shown in SEQ ID NO.14 Vector633 The nucleotide sequence is shown in SEQ ID NO.17; the insert fragment was amplified from plasmid pCW630 using primers phly-LM dal-F shown in SEQ ID NO.15 and primers phly-LM dal-R shown in SEQ ID NO.

16. phly-LM dal , whose nucleotide sequence is shown in SEQ ID NO.18, connected to the vector fragment Vector633 With insert phly-LM dal , and obtain the connection product; (2-B) Transformation of ligation product into E. coli DH5αΔ asd Competent cells were screened by colony PCR using primers pCW633-SX-F shown in SEQ ID NO.19 and primers pCW633-SX-R shown in SEQ ID NO.20 to extract plasmid pCW633, whose 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) Attenuated strain of Listeria monocytogenes LMΔ actAplcB Preparation of competent cells LMΔ actAplcB , the targeting plasmid pCW619-LM dal Electrotransfer into LMΔ actAplcB In competent cells, after electroporation and resuscitation, the strain LMΔ was obtained by colony PCR screening using primers pCW619-Lmdal-F as shown in SEQ ID NO.1 and primers pCW619-R as shown in SEQ ID NO.2, and homologous recombination was performed using primers LMdal-F as shown in SEQ ID NO.3, primers LMdal-R as shown in SEQ ID NO.4, primers HomoLMdal-F as shown in SEQ ID NO.5, and primers HomoLMdal-R as shown in SEQ ID NO.

6. actAplcBdal , prepare LMΔ actAplcBdal Competent cells; (1-2) Insert the targeting plasmid pCW619-LM dat Electrotransfer into LMΔ actAplcBdal In competent cells, after electroporation and resuscitation, the strain LMΔ was obtained by colony PCR screening using primers pCW619-Lmdat-F as shown in SEQ ID NO.7 and primers pCW619-R as shown in SEQ ID NO.2, and homologous recombination was performed using primers LMdat-F as shown in SEQ ID NO.8, primers LMdat-R as shown in SEQ ID NO.9, primers HomoLMdat-F as shown in SEQ ID NO.10, and primers HomoLMdat-R as shown in SEQ ID NO.

11. actAplcBdaldat , prepare LMΔ actAplcBdaldat Competent cells.

3. The method according to claim 1, characterized in that In step (2), strain LMΔ actAplcBdaldat ::pCW633 was prepared as follows: the targeting plasmid pCW633 was electroporated into LMΔ actAplcBdaldat In competent cells, the cells were electroporated and revived, and then cultured. Colony PCR screening was performed using primers asd-SX-F as shown in SEQ ID NO.22 and primers asd-SX-R as shown in SEQ ID NO.23 to obtain strain LMΔ actAplcBdaldat ::pCW633.

4. The method according to claim 1, characterized in that In step (3), LMΔ actAplcBdaldat :: The specific method of inoculating and culturing pCW633 is as follows: strain LMΔ actAplcBdaldat ::pCW633 was streaked onto a BHI plate and cultured at 37°C for 24 h; a single colony was picked from the plate and inoculated into 5 mL of BHI broth and incubated at 37°C, 200 rpm for 16 h; 250 μL of fresh bacterial solution was inoculated into 25 mL of BHI broth and incubated at 37°C, 200 rpm for 16 h; the OD600 value of the bacterial solution was adjusted to 1.0, and the bacterial solution was inoculated into BHI broth at a volume ratio of 1:100 and incubated at 37°C, 200 rpm for 16 h.

5. The method according to claim 1, characterized in that The centrifugal process conditions in step (3) are: 4°C, 12000g for 10 minutes.

6. The method according to claim 1, characterized in that In step (3), the specific method of extraction and purification is as follows: (3-a) The supernatant was filtered through a 0.22 μm filter membrane, concentrated by ultrafiltration, and the precipitate was collected by ultra-high speed centrifugation. The precipitate was resuspended in 0.01 mol / L PBS to obtain a crude product; (3-b) The crude product was treated by ultra-high speed centrifugation using multiple layers of iodixanol solutions of different concentrations. The layers were collected and the liquid layers without flagella were combined. The volume was made up with 0.01 mol / L PBS, and the precipitate was collected by ultra-high speed centrifugation again. The precipitate was resuspended in 0.01 mol / L PBS.

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

8. The method according to claim 6, characterized in that In step (3-b), the mass concentrations of the iodixanol solution are 45%, 35%, 30%, 25%, 20%, 15%, and 10%, which are sequentially layered into the centrifuge tube from bottom to top, with the crude extract layer layered on the top; The process conditions of ultra-high speed centrifugation are: 4°C, 180000g centrifugation for 4 hours; the process conditions of ultra-high speed centrifugation again are: 4°C, 140000g centrifugation for 6 hours.

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

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