A method for improving the production of attenuated listeria monocytogenes membrane vesicles and applications thereof

Abstract

The application relates to a method for improving the yield of attenuated Listeria monocytogenes membrane vesicles and application, wherein, starting from attenuated Listeria monocytogenes LIDeltaAplcB, an LIDeltaAplcBdaldat attenuated strain with dal and dat genes knocked out is constructed, a back-supplement plasmid pCW633 carrying the dal gene is constructed, and the plasmid is electroporated into the attenuated auxotrophic strain LIDeltaAplcBdaldat to construct a stable recombinant strain LIDeltaAplcBdaldat::pCW633. Compared with the wild strain LI, the yield of MVs secreted by the recombinant strain LIDeltaAplcBdaldat::pCW633 can be increased by 1.84 times, and the shape structure, particle size and protein component of the MVs secreted by the recombinant strain are not obviously different from those of the MVs secreted by the wild strain.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method and application for increasing the production of attenuated Listeria ovis membrane vesicles. Background Technology

[0002] Bacterial membrane vesicles (BMVs) are lipid bilayer nanostructures released into the extracellular environment by bacteria during growth. Their diameter ranges from 20 to 200 nm, and they contain various bacterial components, including lipids, proteins, and nucleic acids. 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 classified into Gram-negative bacteria (GMVs) based on the Gram staining characteristics of their source bacteria. - Outer membrane vesicles (OMVs) secreted by Gram-positive bacteria (G) + Membrane vesicles (MVs) secreted by Neisseria meningitidis differ in their secretion mechanisms and structural components due to variations in the cell wall structure and composition of the bacteria from which they originate. The complex and diverse components of BMVs endow them with excellent immunogenicity, effectively stimulating an immune response. Furthermore, their vesicle-like structure endows them with the ability to carry exogenous antigens and encapsulate small molecule drugs; therefore, BMVs are considered a promising vaccine carrier platform or drug delivery system. The marketed group B meningitis vaccine MenBvac is based on Neisseria meningitidis OMVs, demonstrating the broad application prospects of BMVs.

[0003] Currently, most research on BMVs focuses on G - OMVs secreted by bacteria, including Escherichia coli, Neisseria meningitidis, and Pseudomonas aeruginosa, are noteworthy. However, it is important to note that OMVs contain G... - The presence of lipopolysaccharide (LPS), a pyrogen unique to bacterial cell walls, raises questions about the safety of OMV-based biological agents, which to some extent limits their application. G... + The MVs produced by bacteria do not contain the endotoxin LPS, making them safer. Therefore, using G... + Biological agents developed based on bacterial MVs have advantages over OMVs. However, research based on MVs is relatively limited, with only a few Gram-positive bacteria such as Staphylococcus aureus and Listeria monocytogenes being studied. +According to reports, the main limiting factor is G. + The production of bacterial microvessels (MVs) is relatively low. The secretion of MVs is related to the degree of cross-linking of the peptidoglycan layer in the bacterial cell wall.

[0004] Listeria is a type of Gram-positive bacteria. + Non-spore-forming short rod-shaped bacteria, intracellular parasites, have been identified in several species, commonly including Listeria monocytogenes (LI) and Listeria ivanovii (LI). LM and LI can produce various virulence factors such as endothelin and hemolysin, promoting phagocytosis and lysosomal escape, and can simultaneously stimulate cellular and humoral immune responses. MVs produced by LI are similar to other MVs, possessing advantages such as good biocompatibility, high immunogenicity, and the ability to load exogenous antigens; however, they also suffer from low yields. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for increasing the production of Listeria monocytogenes membrane vesicles in sheep, the specific steps of which are as follows:

[0008] (1) Knock out the dal and dat genes of the attenuated Listeria ovis strain LIΔactAplcB to obtain strain LIΔactAplcBdaldat, and prepare LIΔactAplcBdaldat competent cells.

[0009] (2) The targeting plasmid pCW633 was electroporated into LIΔactAplcBdaldat competent cells to obtain strain LIΔactAplcBdaldat::pCW633.

[0010] (3) Cultivate LIΔactAplcBdaldat::pCW633, centrifuge and collect the supernatant, extract and purify to obtain attenuated Listeria ovis membrane vesicles LI MVs.

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

[0012] (1-1) Competent cells of the attenuated Listeria monocytogenes strain LIΔactAplcB were prepared. The targeting plasmid pCW619-LIdal was electroporated into the competent cells of LIΔactAplcB. After electroporation and recovery, the cells were cultured and colony PCR was performed using primers pCW619-LIdal-F (SEQ ID NO.1) and pCW619-R (SEQ ID NO.2). Homologous recombination was performed and colony PCR was performed using primers LIdal-F (SEQ ID NO.3), LIdal-R (SEQ ID NO.4), HomoLIdal-F (SEQ ID NO.5), and HomoLIdal-R (SEQ ID NO.6) to obtain the strain LIΔactAplcBdal. Competent cells of LIΔactAplcBdal were then prepared.

[0013] (1-2) The targeting plasmid pCW619-LIdat was electroporated into LIΔactAplcBdal competent cells. After electroporation and recovery, the cells were cultured and colony PCR was performed using primers pCW619-LIdat-F (SEQ ID NO.7) and pCW619-R (SEQ ID NO.2). Homologous recombination was performed and colony PCR was performed using primers LIdat-F (SEQ ID NO.8), LIdat-R (SEQ ID NO.9), HomoLIdat-F (SEQ ID NO.10), and HomoLIdat-R (SEQ ID NO.11) to obtain the strain LIΔactAplcBdaldat. LIΔactAplcBdaldat competent cells were then 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: pCW619-LIdal Escherichia coli strain is streaked into LB solid medium supplemented with 100 μg / mL ampicillin and cultured at 37℃ for 16 hours; a single colony is picked and inoculated into LB liquid medium supplemented with 100 μg / mL ampicillin and incubated at 37℃ and 200 rpm for 16 hours to extract plasmid pCW619-LIdal.

[0015] As one of the preferred technical solutions, in step (2), the preparation method of strain LIΔactAplcBdaldat::pCW633 is as follows: the targeting plasmid pCW633 is electroporated into LIΔactAplcBdaldat competent cells, and after electroporation and recovery, the cells are cultured. Colony PCR screening is performed using primers asd-SX-F shown in SEQ ID NO.22 and primers asd-SX-R shown in SEQ ID NO.23 to obtain strain LIΔactAplcBdaldat::pCW633.

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

[0017] (2-A) E. coli carrying plasmid pCW630 were cultured, and plasmid pCW630 was extracted. Its nucleotide sequence is shown in SEQ ID NO. 12. Vector fragment Vector633 was amplified from plasmid pCW630 using primers Vector633-F (SEQ ID NO. 13) and Vector633-R (SEQ ID NO. 14). Its nucleotide sequence is shown in SEQ ID NO. 17. Insert fragment phly-LM dal-F (SEQ ID NO. 15) and phly-LM dal-R (SEQ ID NO. 16) was amplified from plasmid pCW630. Its nucleotide sequence is shown in SEQ ID NO. 18. Vector fragment Vector633 and insert fragment phly-LM dal were ligated to obtain the ligation product.

[0018] (2-B) The ligation product was transformed into Escherichia coli DH5αΔasd competent cells. Colony PCR screening was performed using primers pCW633-SX-F (SEQ ID NO.19) and pCW633-SX-R (SEQ ID NO.20). Plasmid pCW633 was extracted, and its nucleotide sequence is shown in SEQ ID NO.21.

[0019] As one of the preferred technical solutions, the specific method for culturing LIΔactAplcBdaldat::pCW633 in step (3) is as follows: streak LIΔactAplcBdaldat::pCW633 onto a BHI plate and incubate at 37°C for 24 hours; pick a single colony and inoculate it into 5 mL of BHI broth, and incubate at 37°C and 200 rpm for 16 hours; take 250 μL of bacterial solution and inoculate it into 25 mL of BHI broth, and incubate at 37°C and 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 and 200 rpm for 16 hours.

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

[0021] As one of the preferred technical solutions, the specific method for extraction and purification in step (3) 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 ultracentrifugation. The precipitate was resuspended in 0.01 mol / L PBS (pH 7.2) to obtain the crude extract.

[0023] (3-b) The crude extract was subjected to ultra-high speed centrifugation with multiple layers of iodixanol solution of different concentrations, and the layers were collected. The non-flagellated liquid layers were combined, and the volume was made up with 0.01 mol / L PBS (pH 7.2). The precipitate was collected by ultra-high speed centrifugation again and resuspended in 0.01 mol / L PBS (pH 7.2) to obtain the final product.

[0024] As one of the further preferred technical solutions, in step (3-a), the molecular weight cutoff of ultrafiltration concentration is 500kDa, and the process conditions for ultra-high speed centrifugation are: 4℃, centrifugation at 140000g for 6 hours.

[0025] As a further preferred technical solution, in step (3-b), the mass concentration of the iodixanol solution is 45%, 35%, 30%, 25%, 20%, 15%, and 10%, and it is sequentially laid into the centrifuge tube from bottom to top, with the crude extract being laid on the top layer.

[0026] As one of the further preferred technical solutions, in step (3-b), the process conditions for ultra-high speed centrifugation are: 4℃, centrifugation at 180000g for 4 hours; the process conditions for ultra-high speed centrifugation again are: 4℃, centrifugation at 140000g for 6 hours.

[0027] This invention also provides applications of the aforementioned methods in basic research related to attenuated LI MVs, specifically including applications in multi-omics studies of attenuated LI MVs, studies of potential biological functions, and formulation preparation using attenuated LI MVs as carriers.

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

[0029] To address the low yield of attenuated Listeria ovis MVs, this invention increases the yield of attenuated L1 MVs by reducing the degree of cross-linking in the peptidoglycan layer of the bacterial cell wall. D-alanine (D-Ala) plays a crucial role in the cross-linking of peptidoglycan layers during bacterial cell wall synthesis. D-Ala synthesis is related to the dal and dat genes. The dal gene controls the synthesis of alanine racemase (Alr), which converts L-alanine to D-Ala, while the dat gene controls the synthesis of D-amino acid aminotransferase (D-AAT), which converts D-glutamate and pyruvate to D-Ala and α-ketoglutarate via transamination. When the dal and dat genes are knocked out of the bacterial genome, the bacteria cannot form a cell wall and cannot grow in media without exogenous D-Ala. Therefore, this invention adds the dal gene to a plasmid and reintroduces it into dal and dat deletion strains. This reduces the degree of cross-linking of the peptidoglycan layer in the bacterial cell wall while maintaining bacterial growth, thereby increasing MV yield. Currently, there are no methods for increasing MV yield related to the dal and dat genes, thus this invention is original.

[0030] This invention starts with attenuated Listeria monocytogenes LIΔactAplcB, constructing an attenuated strain LIΔactAplcBdaldat with the dal and dat genes knocked out. Simultaneously, a complement plasmid pCW633 carrying the dal gene was constructed and electroporated into the attenuated nutrient-deficient strain LIΔactAplcBdaldat, thus constructing a stable recombinant strain LIΔactAplcBdaldat::pCW633. MVs of the recombinant strain were collected using the LI MVs extraction (ultrafiltration concentration-ultracentrifugation) and purification (density gradient centrifugation) protocol established and optimized by the applicant. The total protein concentration of the recombinant MVs was then determined using a BCA kit. The morphology and particle size characteristics of the MVs were analyzed using transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the protein composition of the MVs was analyzed using SDS-PAGE. Compared with the wild-type strain LI, the recombinant strain LIΔactAplcBdaldat::pCW633 can increase the production of MVs by 1.84 times, and the shape, structure, particle size and protein composition of the MVs secreted by the recombinant strain are not significantly different from those of the wild-type strain.

[0031] In summary, this invention can solve the problem of low yield of attenuated LIMVs and can be used for basic research related to attenuated LIMVs, including multi-omics research on attenuated LIMVs (proteomics, lipidomics, metabolomics, etc.), research on the potential biological functions of attenuated LIMVs (pathogenicity, signal transduction, quorum sensing, stress response, etc.), and related formulation applications using attenuated LIMVs as carriers (drug delivery, antigen targeting, regulation of immune response, etc.). Attached Figure Description

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0033] Figure 1 This diagram illustrates the plasmids involved in the preparation of the recombinant strain LIΔactAplcBdaldat::pCW633 in this invention, where a is the targeting plasmid pCW619-LIdal; b is the targeting plasmid pCW619-LIdat; c is plasmid pCW630; and d is plasmid pCW633.

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

[0035] Figure 3The colony PCR verification results of the recombinant strain LIΔactAplcBdaldat::pCW633 prepared for this invention are shown in Figure a. M represents a 250bp DNA marker. In Figure a, 1 and 2 represent the amplification of LI and LIΔactAplcBdaldat::pCW633 using primers HomoLIdal-F / R, respectively. In Figure b, 1 and 2 represent the amplification of LI and LIΔactAplcBdaldat::pCW633 using primers HomoLIdat-F / R, respectively. In Figure c, 1 and 2 represent the amplification of plasmid pCW630 and Escherichia coli pCW633 using primers pCW633-SX-F / R, respectively. In Figure d, 1 and 2 represent the amplification of plasmid pCW633 and LIΔactAplcBdaldat::pCW633 using primers asd-SX-F / R, respectively.

[0036] Figure 4 The in vitro growth curves of wild-type strain LI and recombinant strain LIΔactAplcBdaldat::pCW633 in this invention are shown.

[0037] Figure 5 This is a comparison of the yield of secreted MVs between wild-type strain LI and recombinant strain LIΔactAplcBdaldat::pCW633 in this invention. * indicates P<0.05.

[0038] Figure 6 Transmission electron microscopy images of MVs secreted by wild-type strain LI and recombinant strain LIΔactAplcBdaldat::pCW633 in this invention, used to observe the morphology of MVs; a is MVs secreted by LI; b is MVs secreted by LIΔactAplcBdaldat::pCW633.

[0039] Figure 7 Dynamic light scattering analysis of MVs secreted by wild-type strain LI and recombinant strain LIΔactAplcBdaldat::pCW633 in this invention was performed to characterize the particle size distribution of MVs.

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

[0041] The following specific examples illustrate the implementation 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 embodiments, and various details in this specification can 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 illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0042] Example 1. Construction of LIΔactAplcBdal strain

[0043] 1.1 Preparation of attenuated strain LIΔactAplcB competent cells

[0044] 1.1.1 Strain resuscitation

[0045] A -20℃ attenuated strain LIΔactAplcB (Lei Y,Zhou Y,Zhang Y,Liu S,Tian S,OuQ,Liu T,Huang H,Tang T,Wang CA Listeria ivanovii balanced-lethal system may be a promising antigen carrier for vaccine construction. MicrobBiotechnol.2022Nov;15(11):2831-2844.) was streaked onto a Brain-Heart Infusion Broth (BHI) plate and incubated upside down at 37℃ for 24 h. A single colony was picked and inoculated onto another BHI plate and incubated upside down at 37℃ for another 24 h.

[0046] 1.1.2 Preparation of competent cells

[0047] Pick 3-4 single colonies from the above plate and inoculate them into 15 mL of BHI broth (containing 0.5 mol / L sucrose), incubating at 37°C and 200 rpm for 16 hours. The next morning, inoculate the aforementioned 15 mL of fresh bacterial culture into 250 mL of BHI broth (containing 0.5 mol / L sucrose), incubating at 37°C and 200 rpm. Zero the chamber with BHI broth (containing 0.5 mol / L sucrose) and monitor the OD600 value of the bacterial culture periodically. When the OD600 value reaches 0.4, add penicillin G solution (final concentration 12.5 μg / mL) and continue incubation. When the OD600 value reaches 0.7 or begins to decrease, collect the bacterial cells. Aliquot the bacterial culture into 50 mL centrifuge tubes, centrifuge at 4°C and 13000 rpm for 5 min, and discard the supernatant. Resuspend the bacterial pellet in 20 mL of pre-cooled 0.5 M sucrose solution, centrifuge at 10,000 rpm for 10 min at 4 °C, discard the supernatant, and repeat this step once. Resuspend the bacterial pellet in 300 μL of 0.5 mol / L sucrose, aliquot into 50 μL vials, and store at -80 °C. All bacterial cell collection must be performed on ice.

[0048] 1.2 Preparation of the targeting plasmid pCW619-LIdal (NCBI accession no. MN528129)

[0049] A strain of *E. coli* carrying pCW619-LIdal (Lei Y, Zhou Y, Zhang Y, Liu S, Tian S, OuQ, Liu T, Huang H, Tang T, Wang CA) stored at -20℃ was streaked onto LB (Luria-Bertani) solid medium (hereinafter referred to as LA plates) supplemented with 100 μg / mL ampicillin (Amp) and incubated at 37℃ for 16 hours. A single colony was then inoculated into 5 mL of LB liquid medium (hereinafter referred to as LA broth) supplemented with 100 μg / mL Amp and incubated at 37℃ and 200 rpm for 16 hours. Following the instructions of the Omega plasmid extraction kit, plasmid pCW619-LIdal was extracted and eluted with an appropriate amount of sterile ddH2O. Plasmid concentration and purity were determined using a Nanodrop 2000. A schematic diagram of the targeting plasmid pCW619-LIdal is shown below. Figure 1 a.

[0050] 1.3 Electric Transfer

[0051] Pre-cool 10μL and 200μL pipette tips and electroporation cuvettes at -20℃, and preheat 1000μL pipette tips and BHI broth (containing 1mol / L sucrose) at 37℃. Thaw LIΔactAplcB competent cells and the targeting plasmid pCW619-LIdal on ice. Add 5μL of plasmid solution dropwise to the corresponding 50μL of competent cells, gently mix with a fingertip, and incubate on ice for 5 min. Transfer the mixture to an electroporation cuvette and incubate on ice for 5 min. Electroporate at 1500V for 5ms, remove the cuvette, and incubate on ice for 5 min. Add 700μL of BHI broth (containing 1mol / L sucrose) to the cuvette, mix the cells, and transfer to a 1.5mL EP tube. Recover at 37℃ and 200rpm for 2h. After recovery, centrifuge at 12000rpm for 2 min, discard approximately 600μL of supernatant, and resuspend the bacterial pellet. Spread the entire resuspended bacterial culture onto BHI solid medium (hereinafter referred to as BE3 plate) containing 3 μg / mL erythromycin (Ery), and incubate at 37℃ for 48h-72h.

[0052] 1.4 Screening after electroporation

[0053] Single colonies from BE3 plates were transferred to 20 μL of ddH2O to prepare a bacterial suspension. This suspension was used as a template for colony PCR screening. The screening primers were pCW619-LIdal-F / pCW619-R, where the pCW619-LIdal-F sequence is shown in SEQ ID NO.1 and the pCW619-R sequence is shown in SEQ ID NO.2. Amplification was performed under standard PCR conditions. After amplification, the results were analyzed by agarose gel electrophoresis (1% agarose gel, 90V).

[0054] 1.5 Homologous recombination

[0055] Single colonies successfully transferred by electroporation were streaked onto D-BE3 plates, which are BHI solid medium supplemented with 3 μg / mL erythromycin (Ery) and 200 μg / mL D-alanine (hereinafter referred to as D-BE3 plates), and cultured at 42°C for 3 consecutive subcultures. The bacterial colonies were scraped and inoculated into BHI liquid medium supplemented with 200 μg / mL D-alanine (hereinafter referred to as D-BHI broth), and incubated at 30°C with shaking at 200 rpm for 16 hours, for 6 consecutive subcultures. The 6th generation bacterial culture was serially diluted with 0.01 mol / L PBS (pH 7.2) to a final concentration of 10⁻⁶. -6 Take 100 μL of the diluted solution and spread it onto BHI solid medium (hereinafter referred to as D-BHI plate) with 200 μg / mL D-alanine added, and incubate at 37℃ for 48 h.

[0056] 1.6 Screening

[0057] Single colonies from the D-BHI plates were streaked onto both D-BE3 and D-BHI plates and incubated at 37°C for 24 hours. Single colonies that did not grow on D-BE3 plates but grew on D-BHI plates were selected for colony PCR screening. A small amount of bacterial growth from the D-BHI plate was transferred to 20 μL ddH2O to prepare a bacterial suspension. This suspension was used as a template for colony PCR screening. The screening primers were Lidal-F / R primers and HomoLidal-F / R primers (colony PCR verification results are shown in [link to relevant documentation]). Figure 3 For step a), amplification can be performed under standard PCR conditions. After amplification, analysis is performed by agarose gel electrophoresis (1% agarose gel, 90V). The LIdal-F sequence is shown in SEQ ID NO.3, the LIdal-R sequence is shown in SEQ ID NO.4, the HomoLIdal-F sequence is shown in SEQ ID NO.5, and the HomoLIdal-R sequence is shown in SEQ ID NO.6.

[0058] 1.7 Sequencing

[0059] The amplification products of the HomoLIdal-F / R primers were sent to the company for sequencing. The strain with correct sequencing was named LIΔactAplcBdal and the strain was preserved in a timely manner.

[0060] Example 2. Construction of the LIΔactAplcBdaldat strain

[0061] 2.1 Preparation of LIΔactAplcBdal competent cells

[0062] Prepare LIΔactAplcBdal competent cells according to 1.1 in Example 1. Note that the culture medium used in this step is D-BHI plates and D-BHI broth (containing 0.5 mol / L sucrose).

[0063] 2.2 Preparation of the targeting plasmid pCW619-LIdat (NCBI accession no. MN 528130)

[0064] Referring to section 1.2 of Example 1, the targeting plasmid pCW619-LIdat (Lei Y, Zhou Y, Zhang Y, Liu S, Tian S, Ou Q, Liu T, Huang H, Tang T, Wang CA) was prepared. The plasmid schematic diagram is shown below. (Note: The original text contains some inconsistencies and inconsistencies. A more accurate translation would require the full context.) Figure 1 b.

[0065] 2.3 Electro-rotation

[0066] Referring to section 1.3 of Example 1, the targeting plasmid pCW619-LIdat was electroporated into LIΔactAplcBdal competent cells. After electroporation and recovery, the bacterial suspension was plated onto D-BE3 plates and cultured at 37°C for 48-72 hours.

[0067] 2.4 Screening after electroporation

[0068] Colony PCR screening of single colonies grown on D-BE3 plates was performed according to step 1.4 of Example 1. The screening primers were pCW619-LIdat-F / pCW619-R, where pCW619-LIdat-F is as shown in SEQ ID NO.7 and pCW619-R is as shown in SEQ ID NO.2.

[0069] 2.5 Homologous recombination

[0070] Homologous recombination was performed as described in step 1.5 of Example 1.

[0071] 2.6 Screening

[0072] Single colonies from the D-BHI plates were streaked onto BHI, D-BE3, and D-BHI plates and incubated at 37°C for 24 hours. Single colonies that did not grow on BHI and D-BE3 plates but grew on D-BHI plates were selected for colony PCR screening.

[0073] A small amount of bacterial growth on a D-BHI plate was transferred to 20 μL of ddH2O to prepare a bacterial suspension. This suspension was then used as a template for colony PCR screening. The screening primers were LIdat-F / R and HomoLIdat-F / R (see [link to colony PCR verification results]). Figure 3(b) The LIdat-F sequence is shown in SEQ ID NO.8, the LIdat-R sequence is shown in SEQ ID NO.9, the HomoLIdat-F sequence is shown in SEQ ID NO.10, and the HomoLIdat-R sequence is shown in SEQ ID NO.11.

[0074] 2.7 Sequencing

[0075] The amplification products of the HomoLIdat-F / R primers were sent to the company for sequencing. The strain with correct sequencing was named LIΔactAplcBdaldat and the strain was preserved in a timely manner.

[0076] Example 3. Construction of the complement plasmid pCW633

[0077] 3.1 Preparation of Escherichia coli DH5αΔasd competent cells (CaCl2 method)

[0078] 3.1.1 Strain resuscitation

[0079] Select Escherichia coli DH5αΔasd strain stored at -20℃ and streak it into 5 mL of LB solid medium supplemented with 50 μg / mL diaminopimelic acid (DAP) (hereinafter referred to as DAP-LB plate) and incubate at 37℃ for 16 hours.

[0080] 3.1.2 Preparation of Escherichia coli DH5αΔasd competent cells

[0081] Pick a single colony and inoculate it into 5 mL of LB broth containing 50 μg / mL DAP (hereinafter referred to as DAP-LB broth), and incubate at 37°C and 200 rpm for 16 hours. Inoculate 1 mL of fresh bacterial culture into 50 mL of DAP-LB broth, and incubate at 37°C and 200 rpm until the OD600 value is approximately 0.5, then collect the bacterial cells. Transfer the bacterial culture to a 50 mL centrifuge tube, incubate on ice for 30 min, centrifuge at 4°C and 8000 rpm for 3 min, and discard the supernatant. Resuspend the culture in 10 mL of pre-chilled 0.1 mol / L CaCl2, centrifuge at 4°C and 6000 rpm for 3 min, and discard the supernatant. Resuspend the culture in 1 mL of pre-chilled 0.1 mol / L CaCl2, aliquot into 50 μL vials, and store at -80°C.

[0082] 3.2 Preparation of Carrier Fragments and Inserted Fragments

[0083] 3.2.1 Extraction of plasmid pCW630

[0084] Escherichia coli strains carrying the pCW630 plasmid, stored at -20℃, were streaked onto LB agar plates and incubated at 37℃ for 16 hours to revive the strains. Single colonies were picked and inoculated into 5 mL of LB broth, and incubated at 37℃ and 200 rpm for 16 hours. Plasmid pCW630 was extracted according to the Omega plasmid extraction kit instructions and eluted with 35 μL of sterile ddH2O. The plasmid concentration and purity were determined using a Nanodrop 2000. The sequence of plasmid pCW630 is shown in SEQ ID NO.12, and a schematic diagram of the plasmid is shown below. Figure 1 c.

[0085] 3.2.2 Preparation of Carrier Fragments and Inserted Fragments

[0086] Primers Vector633-F / Vector633-R were used to amplify the vector fragment Vector633 from plasmid pCW630, and primers phly-LM dal-F / R were used to amplify the insert fragment phly-LM dal from plasmid pCW630. Amplification was performed under standard PCR conditions. After amplification, the fragments were separated by agarose gel electrophoresis (1.5% agarose gel, 90V) and purified using the Omega EZNAGel Extraction Kit. The sequences of primers Vector633-F are shown in SEQ ID NO.13, Vector633-R in SEQ ID NO.14, phly-LM dal-F in SEQ ID NO.15, phly-LM dal-R in SEQ ID NO.16, the vector fragment Vector633 in SEQ ID NO.17, and the insert fragment phly-LM dal in SEQ ID NO.18.

[0087] 3.3 Connection Conversion

[0088] 3.3.1 Connection

[0089] Use the ABclonal MultiF Seamless Assembly Mix (RK21020) seamless ligation kit to ligate the vector fragment Vector633 and the insert fragment phly-LM dal. For specific instructions, please refer to the instruction manual.

[0090] 3.3.2 Transformation

[0091] Thaw *E. coli* DH5αΔasd competent cells on ice, add 5 μL of ligation product, gently mix with a fingertip, and incubate on ice for 30 min. Heat shock at 42°C for 45 s, then quickly transfer to ice and incubate for 2 min. Add 700 μL of LB broth and incubate at 37°C and 200 rpm for 1 h. After incubation, centrifuge at 12000 rpm for 2 min, discard approximately 600 μL of supernatant, and resuspend the bacterial pellet. Depending on experimental needs, plate different volumes of the resuspended bacterial solution onto LB agar plates and incubate at 37°C for 16 h.

[0092] 3.4 Colony PCR

[0093] Single colonies grown on LB agar plates were transferred to 20 μL ddH2O to prepare a bacterial suspension. This suspension was used as a template for colony PCR screening. Primers pCW633-SX-F / R were used for colony PCR screening (see [link to colony PCR verification results]). Figure 3 c). The pCW633-SX-F sequence is shown in SEQ ID NO.19, and the pCW633-SX-R sequence is shown in SEQ ID NO.20.

[0094] 3.5 Sequencing

[0095] Single colonies that were verified to be correct by colony PCR were inoculated into LB broth. Plasmids were extracted and sent to a sequencing company for sequencing. The correctly sequenced plasmid was named pCW633 and stored in *E. coli* pCW633. The sequence of plasmid pCW633 is shown in SEQ ID NO.21, and a schematic diagram of the plasmid is shown below. Figure 1 d.

[0096] Example 4. Construction of LIΔactAplcBdaldat::pCW633

[0097] 4.1 Preparation of competent cells using LIΔactAplcBdaldat

[0098] Prepare LIΔactAplcBdaldat competent cells according to 1.1 in Example 1. Note that the culture medium used in this step is D-BHI plates and D-BHI broth (containing 0.5 mol / L sucrose).

[0099] 4.2 Preparation of plasmid pCW633 for refill

[0100] A strain of *E. coli* pCW633 stored at -20℃ was streaked onto an LB agar plate and incubated at 37℃ for 16 hours to revive the strain. A single colony was picked and inoculated into 5 mL of LB broth and incubated at 37℃ and 200 rpm for 16 hours. Plasmid pCW633 was extracted according to the Omega plasmid extraction kit instructions and eluted with 35 μL of sterile ddH2O. The plasmid concentration and purity were determined using a Nanodrop 2000.

[0101] 4.3 Electric Transfer

[0102] Referring to section 1.3 of Example 1, the complement plasmid pCW633 was electroporated into LIΔactAplcBdaldat competent cells. After electroporation and recovery, the bacterial suspension was plated onto BHI plates and incubated at 37°C for 48-72 hours.

[0103] 4.4 Colony PCR

[0104] Colony PCR screening of single colonies grown on BHI plates was performed according to section 1.4 of Example 1. The screening primers were asd-SX-F / R (see section 1.4 for colony PCR verification results). Figure 3 (d). The asd-SX-F sequence is shown in SEQ ID NO.22, and the asd-SX-R sequence is shown in SEQ ID NO.23.

[0105] 4.5 Preservation of microbial strains

[0106] The corrected colony PCR-verified strain was promptly preserved and named LIΔactAplcBdaldat::pCW633.

[0107] Example 5. Determination of growth curve of LIΔactAplcBdaldat::pCW633

[0108] Recombinant bacterial strain LIΔactAplcBdaldat::pCW633, stored at -20℃, was streaked onto a BHI plate and incubated upside down at 37℃ for 24 hours. A single colony was then inoculated into 5 mL of BHI broth and incubated at 37℃ with shaking at 200 rpm for 16 hours. An appropriate amount of bacterial culture was inoculated into 50 mL of BHI broth, and the absorbance value (OD600) at 600 nm was adjusted to 0.06. The culture was incubated at 37℃ with shaking at 200 rpm, and OD600 was measured every 1 hour. A growth curve was plotted based on OD600. The growth curves of LI and LIΔactAplcBdaldat::pCW633 are shown below. Figure 4 As shown, LIΔactAplcBdaldat::pCW633 exhibits a growth trend that is basically consistent with LI.

[0109] Example 6. Extraction, purification, and characterization of MVs.

[0110] 6.1 Large-volume preparation of culture supernatant

[0111] 6.1.1 Strain resuscitation

[0112] A recombinant strain LIΔactAplcBdaldat::pCW633, stored at -20℃, was streaked onto a BHI plate and incubated upside down at 37℃ for 24 hours. Single colonies were then inoculated onto fresh BHI plates and incubated upside down at 37℃ for another 24 hours. 6.1.2 Preparation of large-volume culture supernatant

[0113] A single colony from the BHI plate was inoculated into 5 mL of BHI broth and incubated at 37°C and 200 rpm for 16 hours. 250 μL of fresh bacterial culture was then inoculated into 25 mL of BHI broth and incubated at 37°C and 200 rpm for 16 hours. After adjusting the OD600 value of the bacterial culture to approximately 1.0, 10 mL of the culture was inoculated into 1 L of BHI broth and incubated at 37°C and 200 rpm for 16 hours. The culture was then centrifuged at 12000 g for 10 minutes at 4°C, and the supernatant was collected.

[0114] 6.2MVs extraction and purification

[0115] 6.2.1 Ultrafiltration Concentration - Ultracentrifugation

[0116] The culture supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was concentrated to 35 mL by ultrafiltration using a 500 kDa filter. The concentrate was centrifuged 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) to obtain the crude MVs extract.

[0117] 6.2.2 Density gradient centrifugation purification

[0118] Iodixanol solutions with concentrations of 45%, 35%, 30%, 25%, 20%, 15%, and 10% were sequentially layered (from bottom to top) into 4 mL centrifuge tubes. The crude extract of MVs was then placed on top. The tubes were centrifuged at 180,000 g for 4 h at 4 °C (with minimal acceleration and deceleration). After centrifugation, the samples were collected from each layer. 20 μL of each layer was taken, and an appropriate amount of SDS-PAGE protein loading buffer was added. The proteins were boiled for 10 min to denature them. The density gradient centrifugation effect was observed by SDS-PAGE gel electrophoresis (5% stacking gel, 10% separating gel) and Coomassie Brilliant Blue staining.

[0119] 6.2.3 Washing and Desalination

[0120] Based on the Coomassie brilliant blue staining results, the non-flagellated layers were combined, and the volume was made up with 0.01 mol / L PBS (pH 7.2). The mixture was then centrifuged 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) to obtain the purified MVs product.

[0121] 6.3MVs characterization

[0122] 6.3.1 Protein concentration determination using the BCA method

[0123] Following 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 follows: Figure 5 As shown, the MV yield of LIΔactAplcBdaldat::pCW633 was 564.67 μg / L of culture supernatant, which was 1.84 times that of LI MVs (307.59 μg / L of culture supernatant).

[0124] 5.3.2 TEM characterization of MVs morphological features

[0125] The MVs suspension was dropped onto a 400-mesh copper grid and stained with 2% phosphotungstic acid negative staining. The morphological characteristics of the MVs were characterized under a transmission electron microscope (80 kV). MVs secreted by the wild-type LI strain were observed... Figure 6 See the MVs secreted by a,LIΔactAplcBdaldat::pCW633. Figure 6 b. For example Figure 6 As shown, MVs exhibit a typical spherical structure under TEM with a clean background. Furthermore, the MVs secreted by LIΔactAplcBdaldat::pCW633 show no significant difference in shape, structure, or size from LI.

[0126] 6.3.3 Dynamic light scattering analysis characterizes the particle size characteristics of MVs

[0127] The MV 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 using a Malvern particle size analyzer (Zetasizer Nano ZS). The average particle size of the MVs was reported as the Z-average, and the particle size distribution characteristics of the MVs were visualized using Intensity PSD. The structure for dynamic light scattering analysis is shown below. Figure 7 The average particle size of the MVs secreted by LI is 109.2 nm, and the average particle size of LIΔactAplcBdaldat::pCW633MVs is 118.8 nm, and the particle size distributions of the two are basically the same.

[0128] 6.3.4 SDS-PAGE characterization of MV protein composition

[0129] Take 10 μL of MVs sample, add an appropriate amount of SDS-PAGE protein loading buffer, and boil for 10 min to denature the protein. Prepare SDS-PAGE gels (5% stacking gel, 10% separating gel), load the sample, and perform electrophoresis at a constant voltage of 80V for 30 min, then switch to 120V for 1 h. After electrophoresis, stain with Coomassie Brilliant Blue at room temperature for 1 h, then destain until the background is clean, and image using a gel imaging system. Figure 8 It can be seen that the protein composition of the MVs secreted by LIΔactAplcBdaldat::pCW633 is basically the same as that of the MVs secreted by LI.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method of increasing the yield of attenuated Listeria monocytogenes membrane vesicles, characterized in that, The specific steps are as follows: (1) Knockout of Listeria monocytogenes attenuated strain LIΔ actAplcB of dal , dat Genes, obtained strain LIΔ actAplcBdaldat and prepare LIΔ actAplcBdaldat competent cells; (2) The plasmid pCW633 was electroporated into LIΔ actAplcBdaldat In the competent cells, the strain LIΔ actAplcBdaldat ::pCW633 was obtained. (3) Culturing LIΔ actAplcBdaldat ::pCW633, centrifuging to obtain supernatant, and extracting and purifying to obtain attenuated L. ivanovii membrane vesicles LI MVs; The preparation method of the targeting plasmid pCW633 is as follows: (2-A) E. coli carrying the pCW630 plasmid was cultured, and the plasmid pCW630, the nucleotide sequence of which is shown in SEQ ID NO. 12, was extracted, and a vector fragment was amplified 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; and an insert fragment was amplified 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 the vector fragment Vector633 was ligated to the insert fragment phly-LM dal , to obtain a ligation product; (2-B) The connection product converts E. coli DH5αΔ asd The competent cells were screened by colony PCR using primers pCW633-SX-F (SEQ ID NO. 19) and pCW633-SX-R (SEQ ID NO. 20), and plasmid pCW633 was extracted, the nucleotide sequence of which is shown in SEQ ID NO.

21.

2. The method of claim 1, wherein, The specific method of step (1) is as follows: (1-1) attenuated strain LIΔ of Listeria shettuckii actAplcB The competent LIΔ was prepared actAplcB The targeting plasmid pCW619-LI dal was electro-transformed into LIΔ actAplcB After the electro-transformation recovery, the competent cells were cultured, and colony PCR screening was performed using primers pCW619-LIdal-F as shown in SEQ ID NO. 1 and primers pCW619-R as shown in SEQ ID NO. 2, homologous recombination, and colony PCR screening was performed using primers LIdal-F as shown in SEQ ID NO. 3, primers LIdal-R as shown in SEQ ID NO. 4, primers HomoLIdal-F as shown in SEQ ID NO. 5, and primers HomoLIdal-R as shown in SEQ ID NO. 6, to obtain strain LIΔ actAplcBdal LIΔ was prepared actAplcBdal The competent cells; the targeting plasmid pCW619-LI dal has the NCBI accession number MN528129; (1-2) The targeting plasmid pCW619-LI dat electrotransformation LIΔ actAplcBdal In the competent cells, after electrotransformation recovery, culture, using primers pCW619-LIdat-F as shown in SEQ ID NO. 7, primers pCW619-R as shown in SEQ ID NO. 2 for colony PCR screening, homologous recombination, using primers LIdat-F as shown in SEQ ID NO. 8, primers LIdat-R as shown in SEQ ID NO. 9, primers HomoLIdat-F as shown in SEQ ID NO. 10, primers HomoLIdat-R as shown in SEQ ID NO. 11 for colony PCR screening, to obtain strain LIΔ actAplcBdaldat , preparation of LIΔ actAplcBdaldat competent cells; the targeting plasmid pCW619-LI dat The NCBI accession number of the targeting plasmid pCW619-LI is MN528130.

3. The method of claim 2, wherein, In step (1-1), the targeting plasmid pCW619-LI dal was prepared as follows: pCW619-LI dal was inoculated into LB solid medium supplemented with 100 pg / mL ampicillin and incubated at 37 °C for 16 hours. A single colony was inoculated into LB liquid medium supplemented with 100 pg / mL ampicillin and incubated at 37 °C, 200 rpm for 16 hours. Plasmid pCW619-LI dal was extracted.

4. The method of claim 1, wherein, In step (2), the strain LIΔ actAplcBdaldat The preparation method of pCW633 is as follows: the targeting plasmid pCW633 is electroporated into LIΔ actAplcBdaldat After the electroporation recovery, the culture in the competent cells is screened by colony PCR using primers asasd-SX-F shown in SEQ ID NO. 22 and asd-SX-R shown in SEQ ID NO. 23, to obtain the strain LIΔ actAplcBdaldat ::pCW633.

5. The method of claim 1, wherein, In step (3), LIΔ actAplcBdaldat The specific method for culturing pCW633 is as follows: the strain LIΔ actAplcBdaldat ::pCW633 is streaked on a BHI plate and cultured at 37°C for 24 hours; a single colony is picked and inoculated into 5 mL of BHI broth, which is shaken at 200 rpm at 37°C for 16 hours; 250 μL of the bacterial solution is taken and inoculated into 25 mL of BHI broth, which is shaken at 200 rpm at 37°C for 16 hours; the OD600 value of the bacterial solution is adjusted to 1.0, and the bacterial solution is inoculated into BHI broth at a volume ratio of 1:100, which is shaken at 200 rpm at 37°C for 16 hours.

6. The method of claim 1, wherein, In step (3), the process conditions of centrifugation are as follows: 4℃, 12000g centrifugation for 10 minutes.

7. The method of claim 1, wherein, The specific method of extraction and purification in step (3) is as follows: (3-a) The supernatant is filtered through a 0.22μm filter membrane, concentrated by ultrafiltration, and the precipitate is obtained by ultra-high speed centrifugation, resuspended in 0.01 mol / L PBS to obtain the crude extract product; (3-b) The crude extract product is treated by ultra-high speed centrifugation using multiple layers of different concentrations of iodixanol solution, and the amastigote liquid layer is collected and combined, the volume is made up with 0.01 mol / L PBS, and the precipitate is obtained by ultra-high speed centrifugation again, resuspended in 0.01 mol / L PBS.

8. The method of claim 7, wherein, In step (3-a), the molecular weight cut-off of ultrafiltration concentration is 500kDa, and the process conditions of ultra-high speed centrifugation are as follows: 4℃, 140000g centrifugation for 6 hours; In step (3-b), the mass concentration of iodixanol solution is 45%, 35%, 30%, 25%, 20%, 15%, and 10%, which is sequentially laid in the centrifuge tube from bottom to top, and the crude extract product is laid in the uppermost layer; In step (3-b), the process conditions of ultra-high speed centrifugation are as follows: 4℃, 180000g centrifugation for 4 hours; and the process conditions of ultra-high speed centrifugation again are as follows: 4℃, 140000g centrifugation for 6 hours.

9. The use of the method of any one of claims 1-8 in the preparation of a preparation using attenuated Listeria monocytogenes membrane vesicles as carriers.

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