Attenuated salmonella typhimurium EN-VNP strain as well as preparation method and application thereof

Abstract

The invention discloses an attenuated salmonella typhimurium EN-VNP (Enhanced Natriuretic Peptide) strain as well as a preparation method and application of the attenuated salmonella typhimurium EN-VNP strain. Comprising the following steps: on the basis of a salmonella VNP20009 strain, flagellin genes fliC and fljB, a phosphoethanolamine transferase gene eptA, a 4-amino-4-deoxy-L-arabinose transferase gene arnT and phosphotransferase yeiU are sequentially knocked out or silenced, and a strain is formed after a phosphotransferase gene lpxE is knocked in or overexpressed, so that the attenuated salmonella typhimurium EN-VNP strain is prepared. According to the attenuated salmonella typhimurium EN-VNP with low toxicity and the T cell adjuvant effect and an EN-OMV antigen delivery system prepared from the attenuated salmonella typhimurium EN-VNP, an improved strain does not contain flagellin, an LPS molecular structure is changed, the attenuated salmonella typhimurium EN-VNP has the low toxicity and the T cell adjuvant effect, and normal multiplication culture can be achieved.

Description

Technical Field

[0001] The invention relates to the fields of biological gene engineering technology and vaccine manufacturing, and in particular to an attenuated Salmonella typhimurium EN-VNP and a preparation method and application thereof. Background Art

[0002] OMVs are outer membrane vesicles released by Gram-negative bacteria, mainly containing bacterial outer membrane and periplasmic components. The outer membrane is composed of phospholipids and lipopolysaccharides, with lipopolysaccharides located on the outside of the membrane and interspersed with membrane proteins. The lumen of the vesicle may contain a variety of substances derived from the bacterial periplasm or cytoplasm, such as proteins, RNA or DNA, and peptidoglycan. Because OMVs have been found to induce T cell immune responses, many studies have been conducted with the goal of investigating the potential immunity of OMV components in vivo. The vesicles enter cells using receptor-mediated endocytosis. OMVs can extract surface proteins into APC cells, and their own characteristics determine that they have the function of adjuvants, making them ideal vaccine carriers. To date, the first-generation OMV vaccine Bexsero (Novartis) has been approved for marketing to prevent epidemic cerebrospinal meningitis group B bacterial infection. There are also several OMV-based products focusing on infectious diseases that are in the clinical stage, including pneumonia, meningitis, whooping cough, etc. In addition, OMVs have demonstrated a series of functions in mediating host cell immune responses. The immune properties of OMVs point to the production of protective humoral and cellular immune responses, which can provide a platform for vaccine development. The use of OMVs derived from Escherichia coli and Salmonella as vaccine antigen delivery systems to induce effective immune cell responses has been studied in depth in many literatures.

[0003] Compared with traditional whole pathogen vaccines, subunit vaccines based on single antigens provide more precise targeting and excellent safety. However, this also determines that they have weaker immunogenicity, and adjuvants need to be added to improve the immunogenicity of the antigen and activate the immune response. Unfortunately, there are few clinically applicable adjuvants with high efficiency and low toxicity, and new safe and potent adjuvants are urgently needed. OMV has good biocompatibility and can be taken up by APC (antigen presenting cells). It has the potential to be used as an adjuvant and antigen delivery carrier, but OMV includes a variety of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), flagellin, lipoprotein, DNA, RNA, and peptidoglycan. These components can promote the release of inflammatory cytokines, causing fever, programmed cell death of normal cells, and sepsis, which limits the clinical use of OMV.

[0004] Salmonella is widely distributed in nature and often lives in humans and animals. It belongs to the Enterobacteriaceae family and is a Gram-negative enteric bacillus. The attenuated Salmonella typhimurium VNP20009 is an auxotrophic bacterium in which the pathogenic gene ΔmsbB and the purine ΔpurI gene are knocked out based on the wild-type Salmonella 14028s. It has good tumor targeting and tumor inhibition effects. There are a number of related tumor treatment drugs under clinical research. It is an attenuated Salmonella typhimurium with clinical safety verification. The OMV produced by VNP20009 is expected to be used as an adjuvant and antigen delivery system, but the presence of endotoxin LPS and flagella in the OMV produced by Salmonella VNP20009 still makes it highly toxic and prone to cause sepsis. Using gene editing to eliminate the toxicity caused by endotoxin LPS and flagella is the main strategy for preparing non-toxic OMVs. However, there are many genes related to the expression of endotoxin LPS and flagella, and the mechanism is complex. Figure 1 This is the mechanism, structure and modification diagram of LPS biosynthesis described in Nature Reviews in 2019 (from Nature Reviews Microbiology 2019, 17: 405). Figure 2 It is a structural component and control gene of the flagella of Salmonella typhimurium (from Nature Reviews Microbiology 2008,6:457), Figure 1 and Figure 2 It can be seen that it is very difficult to select appropriate genes for LPS biosynthesis and flagellar structure for editing. In the process of gene knockout, low bacterial survival rate or even bacterial death often occurs, which will also affect the delivery effect. Therefore, how to construct a safe and effective outer membrane vesicle delivery system is of great significance in vaccine research.

[0005] Currently, there is a lack of attenuated Salmonella typhimurium EN-VNP with low toxicity and T cell adjuvant effect, as well as a preparation method and application thereof. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the present invention provides an attenuated Salmonella typhimurium EN-VNP with low toxicity and T cell adjuvant effect and an OMV antigen delivery system prepared therefrom.

[0007] In order to solve the problems of the prior art, the present invention provides the following technical solutions:

[0008] In a first aspect, the present application provides a method for preparing an attenuated Salmonella typhimurium EN-VNP strain;

[0009] In a second aspect, the present application provides an attenuated Salmonella typhimurium EN-VNP strain obtained by a method for preparing an attenuated Salmonella typhimurium EN-VNP strain.

[0010] In a third aspect, the present application provides an outer membrane vesicle formed by a strain.

[0011] In a fourth aspect, the present application provides a method for coupling or encapsulating antigenic proteins in bacterial outer membrane vesicles EN-OMV.

[0012] In a fifth aspect, the present application provides a bacterial outer membrane vesicle EN-OMV antigen delivery system.

[0013] In a sixth aspect, the present application provides an application of a bacterial outer membrane vesicle EN-OMV antigen delivery system in the preparation of a vaccine.

[0014] In a seventh aspect, the present application provides a vaccine preparation comprising an antigen delivery system.

[0015] In a first aspect, the present application provides a method for preparing an attenuated Salmonella typhimurium EN-VNP strain, comprising the following steps: on the basis of the Salmonella VNP20009 strain, the flagellin genes fliC, fljB, the phosphoethanolamine transferase gene eptA, the 4-amino-4-deoxy-L-arabinose transferase gene arnT, the phosphotransferase yeiU are sequentially knocked out or silenced, and the phosphotransferase gene lpxE is knocked in or overexpressed to form a strain, thereby obtaining an attenuated Salmonella typhimurium EN-VNP strain.

[0016] The Salmonella VNP20009 strain used in the present invention is derived from the ATCC strain storage center (ATCC 202165).

[0017] Further, the nucleotide sequence of the gene fliC is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene fliC is shown in SEQ ID NO.2; the nucleotide sequence of the gene fljB is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the gene fljB is shown in SEQ ID NO.4; the nucleotide sequence of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.6; the nucleotide sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the amino acid sequence of the protein encoded by the phosphotransferase gene yeiU is shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO. The amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.11, and the amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.12.

[0018] Furthermore, the nucleotide sequence of the upstream homologous arm of the flagellin gene fliC is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm of the flagellin gene fliC is shown in SEQ ID NO.14; the nucleotide sequence of the upstream homologous arm of the flagellin gene fljB is shown in SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm of the flagellin gene fljB is shown in SEQ ID NO.16; the nucleotide sequence of the upstream homologous arm of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.18.

[0019] Furthermore, the nucleotide sequence of the upstream homologous arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.19, and the nucleotide sequence of the downstream homologous arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.20; the nucleotide sequence of the upstream homologous arm of the knocked-out phosphotransferase yeiU is shown as SEQ ID NO.21, and the nucleotide sequence of the downstream homologous arm of the knocked-out phosphotransferase yeiU is shown as SEQ ID NO.22; the nucleotide sequence of the upstream homologous arm of the knocked-in phosphotransferase gene lpxE is shown as SEQ ID NO.23, and the nucleotide sequence of the downstream homologous arm of the knocked-in phosphotransferase gene lpxE is shown as SEQ ID NO.24.

[0020] The second aspect of the present application provides an attenuated Salmonella typhimurium EN-VNP strain prepared by a method for preparing an attenuated Salmonella typhimurium EN-VNP strain.

[0021] The third aspect of the present application provides an outer membrane vesicle formed by a strain, wherein the outer membrane vesicle has a double-layer membrane structure, and more preferably, the outer membrane vesicle has a diameter of 20-200 nm.

[0022] The outer membrane vesicles of the present invention can be prepared according to conventional methods in the art, such as removing bacteria and bacterial fragments, concentrating the supernatant of bacterial culture fluid, ultracentrifuging and purifying the outer membrane vesicles by similar processes.

[0023] The fourth aspect of the present application provides a bacterial outer membrane vesicle OMV antigen delivery system containing an attenuated Salmonella typhimurium EN-VNP strain.

[0024] The fifth aspect of the present application provides an application of a bacterial outer membrane vesicle EN-OMV antigen delivery system in the preparation of a vaccine, comprising an outer membrane vesicle and a target antigen or nucleic acid.

[0025] The sixth aspect of the present application provides a vaccine preparation comprising an antigen delivery system.

[0026] Furthermore, the antigen protein or nucleic acid is embedded or coupled with the outer membrane vesicle EN-OMV antigen delivery system.

[0027] In the EN-OMV antigen delivery system of the present invention, the outer side of the EN-OMV can be coupled or adsorbed with the target protein antigen or nucleic acid, and the EN-OMV vesicle can also be coated with the target protein antigen or nucleic acid for delivery to cells in the body.

[0028] The antigen protein of the present invention and the outer membrane vesicle EN-OMV antigen delivery system of the present invention can be embedded or coupled according to conventional methods in the art.

[0029] The method for encapsulating or coupling the antigen protein of the present invention with the outer membrane vesicle EN-OMV antigen delivery system of the present invention comprises: activating the carboxyl group of the surface protein of EN-OMV with EDAC, combining with the -NH2 group of amine-PEG3-biotin, and encapsulating biotin on the surface of EN-OMV; then mixing the biotinylated EN-OMV with the target protein and incubating them to obtain the EN-OMV and antigen coupling product.

[0030] The antigen protein of the present invention may be an antigen protein commonly used in the art, such as an antigen protein of a preventive vaccine or an antigen protein of a tumor vaccine.

[0031] Beneficial effects: The attenuated Salmonella typhimurium EN-VNP with low toxicity and T cell adjuvant effect and the prepared EN-OMV antigen delivery system of the present invention make the improved strains free of flagellin and change the LPS molecular structure, have low toxicity and T cell adjuvant effect, and can be normally amplified and cultured, and have important industrial value. It can be used to manufacture preventive vaccines and tumor vaccines, and can achieve injection administration.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] (1) The present invention knocks out or silences the genes fliC, fljB, phosphoethanolamine transferase gene eptA, 4-amino-4-deoxy-L-arabinose transferase gene arnT, phosphotransferase lpxT, and knocks in or overexpresses the phosphotransferase gene lpxE in a specific order, so that the improved strain does not contain flagellin and the LPS molecular structure is changed, has low toxicity and T cell adjuvant effect, can be normally amplified and cultured, and has important industrial value.

[0034] (2) The EN-OMV antigen delivery system prepared by the present invention does not induce antibody production itself, and can only induce extremely low cytokines such as IL-1β, IL-6, and TNF-α, and has good safety; however, it can significantly enhance the antibody concentration of the immunogenic antigen protein, significantly enhance the immune effect of the antigen protein, and has a good adjuvant effect. Therefore, the EN-OMV antigen delivery system of the present invention fully meets the requirements of vaccine adjuvant design and antigen delivery system, and is both safe and effective.

[0035] (3) It can be used to manufacture preventive vaccines and tumor vaccines, and can be administered by injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a diagram of the biosynthesis, structure and modification of the lipopolysaccharide LPS of the present invention. Figure 1 a is that the synthesis of lipid A and the LPS core domain occurs at the cytoplasmic interface of the cytoplasm and the inner membrane. Figure 1b Possible chemical modifications of lipid A in E. coli and Salmonella.

[0037] Figure 2 This is a diagram of the flagellar components of Salmonella typhimurium of the present invention.

[0038] Figure 3 This is the PCR electrophoresis identification diagram of Example 1 of the present invention.

[0039] Figure 4 This is an electron micrograph of the wild type VNP20009 of the present invention.

[0040] Figure 5 The strain EN-VNP obtained after gene knockout of the present invention

[0041] Electron micrograph of (ΔfliCΔfljBΔeptAΔarnTΔyeiU:lpxE).

[0042] Figure 6 This is an electron microscopic image of the EN-OMV vesicles of Example 2 of the present invention.

[0043] Figure 7 This is a graph showing the cytokine expression levels of Example 3 of the present invention.

[0044] Figure 8 This is a graph showing the antibody expression levels of Example 4 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0046] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0047] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0048] In a first aspect, an embodiment of the present application provides a method for preparing an attenuated Salmonella typhimurium EN-VNP strain, comprising the following steps: on the basis of the Salmonella VNP20009 strain, the flagellin genes fliC, fljB, the phosphoethanolamine transferase gene eptA, the 4-amino-4-deoxy-L-arabinose transferase gene arnT, the phosphotransferase yeiU are sequentially knocked out or silenced, and the phosphotransferase gene lpxE is knocked in or overexpressed to form a strain, thereby obtaining an attenuated Salmonella typhimurium EN-VNP strain.

[0049] The Salmonella VNP20009 strain used in the present invention is derived from the ATCC strain storage center (ATCC 202165).

[0050] In some embodiments, the nucleotide sequence of the gene fliC is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene fliC is shown in SEQ ID NO.2; the nucleotide sequence of the gene fljB is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the gene fljB is shown in SEQ ID NO.4; the nucleotide sequence of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.6; the nucleotide sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the amino acid sequence of the protein encoded by the phosphotransferase gene yeiU is shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO. The amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.11, and the amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.12.

[0051] In some embodiments, the nucleotide sequence of the upstream homologous arm of the flagellin gene fliC is shown as SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm of the flagellin gene fliC is shown as SEQ ID NO.14; the nucleotide sequence of the upstream homologous arm of the flagellin gene fljB is shown as SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm of the flagellin gene fljB is shown as SEQ ID NO.16; the nucleotide sequence of the upstream homologous arm of the phosphoethanolamine transferase gene eptA is shown as SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm of the phosphoethanolamine transferase gene eptA is shown as SEQ ID NO.18.

[0052] In some embodiments, the nucleotide sequence of the upstream homologous arm of the knock-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.19, and the nucleotide sequence of the downstream homologous arm of the knock-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.20; the nucleotide sequence of the upstream homologous arm of the knock-out phosphotransferase yeiU is shown as SEQ ID NO.21, and the nucleotide sequence of the downstream homologous arm of the knock-out phosphotransferase yeiU is shown as SEQ ID NO.22; the nucleotide sequence of the upstream homologous arm of the knock-in phosphotransferase gene lpxE is shown as SEQ ID NO.23, and the nucleotide sequence of the downstream homologous arm of the knock-in phosphotransferase gene lpxE is shown as SEQ ID NO.24.

[0053] A second aspect of an embodiment of the present application provides an attenuated Salmonella typhimurium EN-VNP strain obtained by a method for preparing an attenuated Salmonella typhimurium EN-VNP strain.

[0054] A third aspect of an embodiment of the present application provides an outer membrane vesicle formed by a strain, the outer membrane vesicle having a double-layer membrane structure, and the diameter of the outer membrane vesicle is 20-200 nm.

[0055] The outer membrane vesicles of the present invention can be prepared according to conventional methods in the art, such as removing bacteria and bacterial fragments, concentrating the supernatant of bacterial culture fluid, ultracentrifuging and purifying the outer membrane vesicles by similar processes.

[0056] A fourth aspect of the embodiments of the present application provides a bacterial outer membrane vesicle (OMV) antigen delivery system.

[0057] The fifth aspect of the embodiments of the present application provides an application of a bacterial outer membrane vesicle (OMV) antigen delivery system in the preparation of a vaccine.

[0058] A sixth aspect of an embodiment of the present application provides a vaccine preparation comprising an antigen delivery system.

[0059] In some embodiments, the antigen protein or nucleic acid is encapsulated or coupled to the outer membrane vesicle (OMV) antigen delivery system.

[0060] In the OMV antigen delivery system of the present invention, the outer side of the OMV can be coupled or adsorbed with the target protein antigen or nucleic acid, and the inside of the OMV vesicle can also encapsulate the target protein antigen or nucleic acid for delivery to cells in the body.

[0061] The antigen protein of the present invention and the outer membrane vesicle OMV antigen delivery system of the present invention can be embedded or coupled according to conventional methods in the art.

[0062] The method for embedding or coupling antigen protein in the outer membrane vesicle EN-OMV antigen delivery system of the present invention comprises: activating the carboxyl group of the surface protein of EN-OMV with EDAC, combining with the -NH2 group of amine-PEG3-biotin, and encapsulating biotin on the surface of EN-OMV; then mixing the biotinylated EN-OMV with the target protein and incubating them to obtain the EN-OMV and antigen coupling product.

[0063] The antigen protein of the present invention may be an antigen protein commonly used in the art, such as an antigen protein of a preventive vaccine or an antigen protein of a tumor vaccine.

[0064] The Salmonella VNP20009 used in the examples of the present invention is derived from the ATCC strain storage center (ATCC202165).

[0065] Example 1

[0066] Construction of attenuated Salmonella typhimurium EN-VNP

[0067] 1. Construction of ΔfliC strain

[0068] The fliC gene was knocked out using a dual-plasmid-based CRISPR / Cas9 gene editing system. The CRISPR / Cas9 plasmid has Kan resistance and expresses Cas9 protein, and the pTarget plasmid has Amp resistance and carries sgRNA sequence and homologous recombination repair sequence. The sequence of the gene fliC is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0069] The 20 bp sgRNA sequence for targeting was designed as: 5'-AACGAAATCGACCGTGTATC-3', the upstream homology arm sequence of the knockout gene fliC was shown in SEQ ID NO.13, and the downstream homology arm sequence was shown in SEQ ID NO.14.

[0070] Preparation of VNP containing CRISPR / Cas9 plasmid: VNP monoclonal was inoculated into 2 mL of LB medium and cultured at 37°C until OD 600 =0.6, collect the cells by centrifugation, wash the cells three times with pre-cooled 10% glycerol, and resuspend them with 100 μL of 10% glycerol. Add 1 μg of Cas9 plasmid to competent cells for electroporation, and set the conditions as: 2400V, 200Ω. Then, plate the cells, culture at 30°C, and culture with Kan resistance. After overnight culture, pick a single clone, which is VNP-cas9.

[0071] Preparation of VNP-cas9 strain containing pTarget plasmid: Inoculate VNP-cas9 monoclonal clone into 2 ml LB medium and culture at 30°C until OD 600 =0.1-0.2, add arabinose induction, concentration 3mg / mL, incubate for 1hr, collect the cells by centrifugation, wash 3 times with 10% glycerol, and resuspend with 100μL of 10% glycerol. Add 1μg of pTarget plasmid to the competent cells for electroporation. Then spread on LB plates, Kan+ and Amp+, incubate for more than 16hr, pick single clones, and amplify and culture.

[0072] Remove single plasmid: Add IPTG to the culture medium, culture overnight, pass 2 generations, remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick a single clone to culture and pass down to save the seeds, which is VNPΔfliC.

[0073] 2. Construction of ΔfljB strain based on VNPΔfliC

[0074] The sequence of gene fljB is shown in SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.4.

[0075] The 20 bp sgRNA sequence for targeting was designed as: 5'-GTTTACGGTATTGCCCAGGT-3', the upstream homology arm sequence of the knockout gene fljB was shown in SEQ ID NO.15, and the downstream homology arm sequence was shown in SEQ ID NO.16.

[0076] Preparation of VNPΔfliC strain containing pTarget plasmid: VNPΔfliC single clone (containing cas9 plasmid, Kan resistance) was inoculated into 2 mL of LB medium and cultured at 30°C until OD 600=0.1-0.2, add arabinose induction, concentration 3mg / mL, incubate for 1hr, collect the cells by centrifugation, wash 3 times with 10% glycerol, and resuspend with 100μL of 10% glycerol. Add 1μg of pTarget plasmid to the competent cells for electroporation. Then spread on LB plates, Kan+ and Amp+, incubate for more than 16hr, pick single clones, and amplify and culture.

[0077] Remove single plasmid: Add IPTG to the culture medium, culture overnight, pass 2 generations, remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick a single clone to culture and pass down to save the seeds, which is VNPΔfliCΔfljB.

[0078] 3. Knockout of eptA gene based on VNPΔfliCΔfljB

[0079] The sequence of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.6.

[0080] The 20 bp sgRNA sequence for targeting was designed as: 5'-GGCGAATCATTGGGTGAAAA-3', the upstream homology arm sequence for knocking out the phosphoethanolamine transferase gene eptA was shown in SEQ ID NO.17, and the downstream homology arm sequence was shown in SEQ ID NO.18.

[0081] Preparation of VNPΔfliCΔfljB strain containing pTarget plasmid: VNPΔfliCΔfljB single clone (containing cas9 plasmid, Kan resistance) was inoculated into 2 ml LB medium and cultured at 30°C until OD 600 =0.1-0.2, add arabinose induction, concentration 3mg / mL, incubate for 1hr, collect the cells by centrifugation, wash 3 times with 10% glycerol, and resuspend with 100μL of 10% glycerol. Add 1μg of pTarget plasmid to the competent cells for electroporation. Then spread on LB plates, Kan+ and Amp+, incubate for more than 16hr, pick single clones, and amplify and culture.

[0082] Remove single plasmid: Add IPTG to the culture medium, culture overnight, pass 2 generations, remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick a single clone to culture and pass down to save the seeds, which is VNPΔfliCΔfljBΔeptA.

[0083] 4. Knockout of arnT gene based on VNPΔfliCΔfljBΔeptA

[0084] The sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.8.

[0085] The 20bp sgRNA sequence for targeting was designed as: 5'-TCAACAGCCGCCTGCTCTGG-3', the upstream homology arm sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT knockout was shown in SEQ ID NO.19, and the downstream homology arm sequence was shown in SEQ ID NO.20.

[0086] Preparation of VNPΔfliCΔfljBΔeptA strain containing pTarget plasmid: VNPΔfliCΔfljBΔeptA single clone (containing cas9 plasmid, Kan resistance) was inoculated into 2 mL of LB medium and cultured at 30°C until OD 600 =0.1-0.2, add arabinose induction, concentration 3mg / mL, incubate for 1hr, collect the cells by centrifugation, wash 3 times with 10% glycerol, and resuspend with 100μL of 10% glycerol. Add 1μg of pTarget plasmid to the competent cells for electroporation. Then spread on LB plates, Kan+ and Amp+, incubate for more than 16hr, pick single clones, and amplify and culture.

[0087] Remove a single plasmid: Add IPTG to the culture medium, culture overnight, pass 2 generations, remove the pTarget plasmid. At this time, VNP loses its Amp resistance. Pick a single clone to culture and pass down to save the seeds, which is VNPΔfliCΔfljBΔeptAΔarnT.

[0088] 5. Knockout of yeiU gene based on VNPΔFliCΔFljBΔeptAΔarnT

[0089] The sequence of the phosphotransferase gene yeiU is shown as SEQ ID NO.9, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.10.

[0090] The 20bp sgRNA sequence for targeting was designed as: 5'-TGCGCAGCGAGTTTAACGGT-3', the upstream homology arm sequence for knocking out the phosphotransferase gene yeiU was shown in SEQ ID NO.21, and the downstream homology arm sequence was shown in SEQ ID NO.22.

[0091] Preparation of VNPΔFliCΔFljBΔeptAΔarnT strain containing pTarget plasmid: VNPΔFliCΔFljBΔeptAΔarnT single clone (containing cas9 plasmid, Kan resistance) was inoculated into 2 mL of LB medium and cultured at 30°C until OD600 =0.1-0.2, add arabinose induction, concentration 3mg / mL, incubate for 1hr, collect the cells by centrifugation, wash 3 times with 10% glycerol, and resuspend with 100μL of 10% glycerol. Add 1μg of pTarget plasmid to the competent cells for electroporation. Then spread on LB plates, Kan+ and Amp+, incubate for more than 16hr, pick single clones, and amplify and culture.

[0092] Remove single plasmid: Add IPTG to the culture medium, culture overnight, pass 2 generations, remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick a single clone to culture and pass down to save the seeds, which is VNPΔfliCΔfljBΔeptAΔarnTΔyeiU.

[0093] 6. Knock-in the lpxE gene based on VNPΔfliCΔfljBΔeptAΔarnTΔyeiU

[0094] The sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO.11, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.12.

[0095] The 20 bp sgRNA sequence for targeting was designed as: 5'-ACATCAAGTTGTAATTGATA-3', and the upstream homology arm sequence of the knock-in phosphotransferase gene lpxE was shown in SEQ ID NO.23, and the downstream homology arm sequence was shown in SEQ ID NO.24.

[0096] Preparation of VNPΔfliCΔfljBΔeptAΔarnTΔyeiU strain containing pTarget plasmid: VNPΔfliCΔfljBΔeptAΔarnTΔyeiU single clone (containing cas9 plasmid, Kan resistance) was inoculated into 2 mL of LB medium and cultured at 30°C until OD 600 =0.1-0.2, add arabinose induction, concentration 3mg / mL, incubate for 1hr, collect the cells by centrifugation, wash 3 times with 10% glycerol, and resuspend with 100μL of 10% glycerol. Add 1μg of pTarget plasmid to the competent cells for electroporation. Then spread on LB plates, Kan+ and Amp+, incubate for more than 16hr, pick single clones, and amplify and culture.

[0097] Remove the double plasmids: add IPTG to the culture medium, culture overnight, pass 2 generations, remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick a single clone for culture and pass it. Increase the culture temperature to 37°C overnight, remove the CRISPR / Cas9 plasmid, and save the seeds, which is VNPΔfliCΔfljBΔeptAΔarnTΔyeiU:lpxE.

[0098] The strain obtained by knocking out the flagellin genes fliC and fljB, the phosphoethanolamine transferase gene eptA, the 4-amino-4-deoxy-L-arabinose transferase gene arnT, the phosphotransferase yeiU, and knocking in or overexpressing the phosphotransferase gene lpxE was named EN-VNP strain.

[0099] 7. Identify eptA gene knockout using the following primers:

[0100] Upstream primer: 5'-tgccttgagcatcaaccgtg-3'

[0101] Downstream primer: 5'-tcggcgtgctgatgactatc-3'

[0102] The PCR sequencing of the wild strain sequence range is: 4440726-4443344, theoretically a total of 2618nt; the actual sequencing result of the mutant strain is 974nt, and the sequence is shown in SEQ ID NO.25.

[0103] 8. Identify arnT gene knockout using the following primers:

[0104] Upstream primer: 5'-ATATTGCGCGCGGGCATAACG-3'

[0105] Downstream primer: 5'-AGACTCAGTAGCGCATAGGC-3'

[0106] The sequence range of the wild strain by PCR sequencing is: 2350620-2353357, theoretically a total of 2737nt; the actual sequencing result of the mutant strain is 1092nt, and the sequence is shown in SEQ ID NO.26.

[0107] 9. Identify the knockout of yeiU gene using the following primers:

[0108] Upstream primer: 5'-gatcgtcaactgattcatgc-3'

[0109] Downstream primer: 5'-tcggttgagatttgaccatg-3'

[0110] The PCR sequencing of the wild strain sequence range is: 2254197-2256563, theoretically a total of 1646 nt; the actual sequencing result of the mutant strain is 926 nt, and the sequence is shown in SEQ ID NO.27.

[0111] 10. Identify the knock-in of lpxE gene using the following primers:

[0112] Upstream primer: 5'-cgctgagtgccattgcgtca-3'

[0113] Downstream primer: 5'-ctgctctttggtggtcgcga-3'

[0114] The sequence range of the wild strain by PCR sequencing is: 1949168-1951780, theoretically 2612 nt; the actual sequencing result of the mutant strain is 1990 nt, and the sequence is shown in SEQ ID NO.28.

[0115] PCR electrophoresis identification diagram Figure 3 As shown, eptA, arnT, yeiU have been successfully knocked out and lpxE has been successfully knocked in. Through electron microscopy observation, Figure 4 As can be seen, the VNP20009 wild type has flagella visible under electron microscopy ( Figure 4 ), strain EN-VNP (VNP VNPΔfliCΔfljBΔeptAΔarnTΔyeiU:lpxE) obtained after gene knockout, electron microscopy photos show that flagella are knocked out ( Figure 5 ).

[0116] Example 2

[0117] Extraction, Preparation and Characterization of Outer Membrane Vesicles (OMV)

[0118] The strain EN-VNP obtained in Example 1 was expanded and cultured, and the seed solution was inoculated into a fermentation basal medium (ratio: 10 g / L plant peptone, 5 g / L yeast extract, 10 g / L sodium chloride) at a ratio of 1:100 (volume ratio). The fermentation parameters were set at pH 7.0, rotation speed 220 rpm, and temperature 37°C.

[0119] During the fermentation process, OD was measured every 1 hour. 600 Value, OD600 is about 1 at 8 hours of fermentation, centrifuge at 5000rpm for 30min, remove bacteria and cell debris, and collect the culture supernatant. The culture supernatant is filtered through a 0.22μm filter membrane and concentrated with a 100Kda ultrafilter to collect the supernatant. Nuclease is then added to remove nucleic acid impurities, and the pure OMV is collected by centrifugation at 100000rpm for 2h, which is the OMV vesicle (sphere) used in the delivery system. Conventional embedding and coupling can be performed to deliver antigens later. Samples are taken for electron microscopy for characterization. Figure 6 As shown, the diameter is 20-200nm.

[0120] Example 3

[0121] Coupling of antigenic proteins to EN-OMV outer membrane vesicles

[0122] Method for encapsulating biotin on the surface of EN-OMV: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) can activate the carboxyl group of EN-OMV surface protein and bind it to the -NH2 group of amine-PEG3-biotin to form an amide bond. In phosphate buffered saline, the biotin derivative Amine-PEG3-biotin, EDAC and EMV were incubated at 4°C for 2 hours, and Amine-PEG3-biotin was coated on the surface of EN-OMV (biotinylated EN-OMV). The 100kDa cut-off membrane ultrafiltration tube was washed to eliminate unbound Amine-PEG3-biotin and EDAC.

[0123] The avidin protein and the antigen protein (such as RFP protein) are connected through a flexible linker to form the target protein: the biotinylated EN-OMV and the target protein are mixed at a ratio of 2:1 (mass / mass) and incubated at 4°C for 2 hours. Ultracentrifuge at 100,000×g and 4°C for 2 hours. The obtained particles are washed twice with PBS and finally resuspended in PBS buffer to obtain the EN-OMV and antigen coupling product.

[0124] Test Example 1

[0125] Immune cell inflammatory factor release test

[0126] Raw246.7 macrophages were cultured in DMEM medium supplemented with 10% complement inactivated serum, and macrophages were stimulated with OMV prepared in Examples 2 and 3 for 4 hours. The control group was PBS solution, and then the cells were lysed with TRIZOI to extract RNA, and the RNA was reverse transcribed into cDNA. The expression of inflammatory factors was detected by Q-PCR, and the expression level of inflammatory factors was calculated by the ΔΔct method. The results are shown in Figure 7 As shown, the expression levels of inflammatory factors IL-1β, IL-6 and TNF-α were significantly decreased.

[0127] Test Example 2

[0128] Humoral immune response test

[0129] The EN-OMV prepared in Example 3 was coupled to RFP protein, and the EN-OMV-RFP conjugate was used to immunize mice on the 1st and 14th days. The control groups were PBS solution, RFP solution, and EN-OMV solution, respectively. The mouse serum was collected on the 28th day, and the RFP-specific antibody concentration in the serum was detected by ELISA. The results are shown in Figure 8As shown, RFP is a commonly used tool protein in cell biology, and its immunogenicity is low, which is one of the reasons why it is widely used as a tool for cell biology research. Compared with the RFP protein antigen, the antibody concentration of the EN-OMV-RFP immune group increased significantly (increased by more than 8 times). At the same time, the antibody concentration produced by EN-OMV itself is almost exactly the same as that of PBS, which shows that EN-OMV itself has low immunogenicity, but has a good adjuvant effect, which significantly enhances the immune effect of RFP protein. In sharp contrast to Experimental Example 1, although the inflammatory factors such as IL-1β, IL-6, and TNF-α produced by EN-OMV in Experimental Example 1 are significantly reduced, showing good safety; However, in Experimental Example 2, it can produce a good antibody concentration. It can be seen that the EN-OMV prepared in Example 3 fully meets the requirements of vaccine adjuvant design and antigen delivery system, which is both safe and effective.

[0130] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A method for preparing an attenuated Salmonella typhimurium EN-VNP strain, characterized in that The method comprises the following steps: on the basis of the Salmonella VNP20009 strain, flagellin genes fliC and fljB, phosphoethanolamine transferase gene eptA, 4-amino-4-deoxy-L-arabinose transferase gene arnT, phosphotransferase yeiU are sequentially knocked out or silenced, and the phosphotransferase gene lpxE is knocked in or overexpressed to form a strain, thereby preparing an attenuated Salmonella typhimurium EN-VNP strain.

2. The method for preparing the attenuated Salmonella typhimurium EN-VNP strain according to claim 1, characterized in that: The nucleotide sequence of the gene fliC is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene fliC is shown in SEQ ID NO.2; the nucleotide sequence of the gene fljB is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the gene fljB is shown in SEQ ID NO.4; the nucleotide sequence of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.6; the nucleotide sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the amino acid sequence of the protein encoded by the phosphotransferase gene yeiU is shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO. As shown in SEQ ID NO.11, the amino acid sequence of the protein encoded by the phosphotransferase gene lpxE is shown in SEQ ID NO.

12.

3. The method for preparing the attenuated Salmonella typhimurium EN-VNP strain according to claim 1, characterized in that: The nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fliC is shown as SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fliC is shown as SEQ ID NO.14; the nucleotide sequence of the upstream homologous arm of the knockout flagellin gene fljB is shown as SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm of the knockout flagellin gene fljB is shown as SEQ ID NO.16; the nucleotide sequence of the upstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown as SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm of the knockout phosphoethanolamine transferase gene eptA is shown as SEQ ID NO.

18.

4. The method for preparing the attenuated Salmonella typhimurium EN-VNP strain according to claim 1, characterized in that: The nucleotide sequence of the upstream homologous arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.19, and the nucleotide sequence of the downstream homologous arm of the knocked-out 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown as SEQ ID NO.20; the nucleotide sequence of the upstream homologous arm of the knocked-out phosphotransferase yeiU is shown as SEQ ID NO.21, and the nucleotide sequence of the downstream homologous arm of the knocked-out phosphotransferase yeiU is shown as SEQ ID NO.22; the nucleotide sequence of the upstream homologous arm of the knocked-in phosphotransferase gene lpxE is shown as SEQ ID NO.23, and the nucleotide sequence of the downstream homologous arm of the knocked-in phosphotransferase gene lpxE is shown as SEQ ID NO.

24.

5. The attenuated Salmonella typhimurium EN-VNP strain obtained by the preparation method of the attenuated Salmonella typhimurium EN-VNP strain according to claim 1.

6. The outer membrane vesicle formed by the strain according to claim 5, characterized in that: The outer membrane vesicle has a double-layer membrane structure, and the diameter of the outer membrane vesicle is 20-200 nm.

7. A bacterial outer membrane vesicle (OMV) antigen delivery system containing the attenuated Salmonella typhimurium EN-VNP strain according to claim 5.

8. Use of the bacterial outer membrane vesicle (OMV) antigen delivery system according to claim 7 in the preparation of a vaccine, characterized in that: The outer membrane vesicles according to claim 6 are used to load and deliver target antigens or nucleic acids.

9. A vaccine formulation comprising the antigen delivery system of claim 8.

10. The vaccine preparation according to claim 9, characterized in that: The antigen protein or nucleic acid is embedded or coupled with the outer membrane vesicle EN-OMV antigen delivery system.

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