Salmonella typhimurium EN-VNP strain based on flagellin and LPS attenuation as well as preparation method and application of salmonella typhimurium EN-VNP strain

Abstract

The invention discloses a salmonella typhimurium EN-VNP strain based on flagellin and LPS attenuation as well as a preparation method and application of the salmonella typhimurium EN-VNP strain, and particularly relates to the technical field of biology. The bacterial strain is formed by sequentially knocking out or silencing flagellin genes fliC and fljB, a phosphoethanolamine transferase gene eptA, a 4-amino-4-deoxy-L-arabinose transferase gene arnT and phosphotransferase yeiU on the basis of a salmonella VNP 20009 bacterial strain, and knocking in or overexpressing a phosphotransferase gene lpxE. The salmonella typhimurium EN-VNP strain disclosed by the invention has relatively high safety, the attenuated strain does not contain flagellin, and a lipid A molecule of LPS only retains one phosphate group, so that the safety of the strain is improved, and the strain is more suitable for serving as a vaccine and a drug delivery carrier.

Description

Technical Field

[0001] The present invention relates to the fields of biological genetic engineering technology and vaccine manufacturing, and particularly relates to an attenuated Salmonella typhimurium EN-VNP strain based on flagellin and LPS, a preparation method thereof, and applications in the fields of vaccines / tumor therapy. Background Art

[0002] VNP 20009 is a Gram-negative facultative anaerobe that can grow and replicate within host cells. VNP 20009 is a auxotrophic strain that lacks the pathogenic gene ΔmsbB and the purine synthesis gene ΔpurI based on the wild-type Salmonella typhimurium 14028s, and is expected to be used as a preventive vaccine against Salmonella typhimurium infection or a tumor therapy agent. However, there are a large number of flagella and endotoxin LPS in Salmonella VNP 20009, making it still highly toxic, easily causing inflammation and swelling of immune organs and even sepsis. Flagellin is the protein subunit of bacterial flagella, composed of conserved N-terminal and C-terminal D0-D1 domains separated by a hypervariable region, and is a ligand of Toll-like receptor 5 (TLR-5). TLR-5 is expressed on various cells such as epithelial cells, monocytes, and immature dendritic cells. Flagellin can induce the expression of various pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Endotoxin (LPS) is a ligand of Toll-like receptor 4 (TLR4), which binds to the TLR4-medullary differentiation factor 2 (MD-2) complex to activate a series of signal cascades that promote inflammatory responses. Using gene editing to eliminate the toxicity caused by flagella and endotoxin LPS is the main strategy for preparing non-toxic Salmonella. And there are many genes related to the expression of endotoxin LPS and flagella, and the mechanism is complex. Figure 1 is the flagellar structural component and control gene of Salmonella typhimurium (from Nature Reviews Microbiology Volume 6 June 2008 457), Figure 2 is the mechanism, structure, and modification diagram of LPS biosynthesis described in "Nature Reviews" in 2019 (from Nature Reviews Microbiology Reviews Volume 17 July 2019 407), by Figure 1 and Figure 2 It can be seen that it is extremely difficult to select appropriate genes related to LPS biosynthesis and flagellar structure for editing. During the gene knockout process, there often occur phenomena such as low bacterial survival rate or even bacterial death, 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. Summary of the Invention

[0003] In view of the deficiencies of the above-mentioned existing technologies, the present application provides a flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain, its preparation method and applications. The attenuated Salmonella has high safety.

[0004] To achieve the above application objectives, the technical solutions adopted in the present application are as follows:

[0005] In the first aspect, the present application provides a flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain;

[0006] In the second aspect, the present application provides a preparation method of a flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain;

[0007] In the third aspect, the present application provides a method using the attenuated Salmonella EN-VNP strain as a delivery vector;

[0008] In the fourth aspect, the present application provides a method for detecting the gene knockout or silencing effect in a flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain.

[0009] In the fifth aspect, the present application provides an application of a flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain in the preparation of vaccine preparations.

[0010] In the sixth aspect, the present application provides an application of a flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain in the preparation of anti-tumor drugs.

[0011] The flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain provided in the first aspect of the present application is a strain formed by sequentially knocking out or silencing the flagellin genes fliC, fljB, phosphoethanolamine transferase gene eptA, 4-amino-4-deoxy-L-arabinose transferase gene arnT, phosphotransferase yeiU on the basis of the Salmonella VNP 20009 strain and knocking in or overexpressing the phosphotransferase gene lpxE.

[0012] The Salmonella VNP 20009 strain used in the present invention is derived from the ATCC strain preservation center (ATCC202165).

[0013] Further, the nucleotide 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; the nucleotide sequence of the gene fljB is shown in SEQ ID NO.3, and the nucleotide sequence of the encoded protein 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 nucleotide sequence of the encoded protein 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 nucleotide sequence of the encoded protein is shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the nucleotide sequence of the encoded protein is shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO.11, and the nucleotide sequence of the encoded protein is shown in SEQ ID NO.12.

[0014] A preparation method of a flagellin and LPS attenuated Salmonella typhimurium EN-VNP strain provided by the second aspect of the present application includes the following steps:

[0015] (1) Construct a ΔfliC strain, knockout the fliC gene through the CRISPR / Cas9 gene editing system, and use the designed sgRNA sequence and homologous arm sequence for gene knockout;

[0016] (2) On the basis of the ΔfliC strain, construct a ΔfljB strain, knockout the fljB gene through the CRISPR / Cas9 system, and use the designed sgRNA sequence and homologous arm sequence for gene knockout;

[0017] (3) Knockout the eptA gene on the basis of the ΔfliCΔfljB strain, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout;

[0018] (4) Knockout the arnT gene on the basis of the ΔfliCΔfljBΔeptA strain, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout;

[0019] (5) Knockout the yeiU gene on the basis of the ΔfliCΔfljBΔeptAΔarnT strain, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout;

[0020] (6) Overexpress the lpxE gene in the ΔfliCΔfljBΔeptAΔarnTΔyeiU strain, and use a plasmid containing the J23100 constitutive promoter and Kan+ resistance as the expression vector for the lpxE gene for gene overexpression.

[0021] Further, in step (1), the sgRNA sequence targets 5’-AACGAAATCGACCGTGTATC-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.14;

[0022] In step (2), the sgRNA sequence targets 5’-GTTTACGGTATTGCCCAGGT-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.15, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.16;

[0023] In step (3), the sgRNA sequence targets 5’-GGCGAATCATTGGGTGAAAA-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.18;

[0024] In step (4), the sgRNA sequence targets 5’-TCAACAGCCGCCTGCTCTGG-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.19, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.20;

[0025] In step (5), the sgRNA sequence targets 5’-TGCGCAGCGAGTTTAACGGT-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.21, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.22;

[0026] In step (6), use a plasmid containing the J23100 constitutive promoter and Kan+ resistance as the expression vector J23-lpxE for the lpxE gene.

[0027] Further, the culture conditions of the strain are 37 °C to OD600 = 0.6 - 0.8; the electrotransformation conditions are set to 2400V, 200Ω; use arabinose to induce the expression of Cas9 protein, with a concentration of 3mg / mL and an incubation time of 1 hour; remove the pTarget plasmid by IPTG induction, culture overnight and passage 2 times to lose Amp resistance.

[0028] The application provided by the fifth aspect of the present application, which is based on the attenuated Salmonella typhimurium EN-VNP strain with flagellin and LPS, can activate the immune system.

[0029] The application of the attenuated Salmonella typhimurium EN-VNP strain with flagellin and LPS provided by the sixth aspect of the present application in the preparation of vaccine preparations can activate the immune system and exert the immune activation effect of the vaccine.

[0030] The application of the attenuated Salmonella typhimurium EN-VNP strain with flagellin and LPS provided by the seventh aspect of the present application in the preparation of anti-tumor drugs can activate the immune system and exert the immune activation effect of activating anti-tumor immunity.

[0031] Beneficial effects: By knocking out or silencing the genes related to the synthesis of flagellin and endotoxin LPS, the present invention successfully constructed an attenuated Salmonella typhimurium EN-VNP strain based on flagellin and LPS, significantly reducing its toxicity and reducing the risk of immune organ inflammation and septicemia. The present invention provides a novel method for attenuating the Salmonella typhimurium VNP 20009 strain, achieving the attenuation of the strain through gene knockout and overexpression in a specific order.

[0032] Compared with the prior art, the present invention has the following advantages: (1) Improved safety: The attenuated strain does not contain flagellin and only one phosphate group is retained in the lipid A molecule of LPS, improving the safety of the strain and making it more suitable as a vaccine and drug delivery carrier.

[0033] (2) Industrial application value: Since the attenuated Salmonella EN-VNP strain has high safety and can be normally amplified and cultured, it has important industrial application value and can be used for vaccine manufacturing and the development of anti-tumor drugs.

[0034] (3) Tumor treatment potential: The attenuated Salmonella EN-VNP strain can be used for tumor treatment or as a delivery carrier for tumor treatment drugs to achieve injection administration, providing a new strategy for tumor treatment. Reduced inflammatory response: In experiments, compared with the wild-type Salmonella VNP 20009, the levels of inflammatory factors IL-6 and TNF-α induced by the EN-VNP strain decreased significantly, indicating that the inflammatory response it caused in vivo was relatively mild.

[0035] (4) Precision of gene editing: Through the CRISPR / Cas9 gene editing technology, the present invention can accurately knock out or silence target genes, improving the precision and efficiency of gene editing.

[0036] (5) The present invention not only provides an attenuated Salmonella strain, but also offers new ideas and methods for the development of vaccines and anti-tumor drugs. Experimental verification: Through electron microscopy observation and inflammatory factor concentration detection tests, the present invention provides experimental data to support its attenuation effect and safety, increasing the credibility of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following-described drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is a diagram of the flagellar assembly of Salmonella typhimurium of the present invention.

[0039] Figure 2 It is a diagram of the biosynthesis, structure, and modification of lipopolysaccharide (LPS) of the present invention. Figure 2 a The synthesis of lipid A and the LPS core domain occurs at the cytoplasmic interface of the cytoplasm and the inner membrane. Figure 2 b Possible chemical modifications of Escherichia coli and Salmonella lipid A.

[0040] Figure 3 It is a PCR electrophoresis identification diagram of Example 1 of the present invention;

[0041] Figure 4 It is an electron micrograph of the wild type VNP 20009 of the present invention;

[0042] Figure 5 It is an electron micrograph of the strain EN-VNP (ΔfliCΔfljBΔeptAΔarnTΔyeiU:lpxE) obtained after gene knockout and overexpression of the present invention;

[0043] Figure 6 It is a diagram of the serum inflammatory factor concentration in Test Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0044] In order to make the technical problems to be solved, technical solutions, and beneficial effects of the present application more clearly understood, the following further details the present application in combination with 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.

[0045] In this application, the term "and / or" describes the association relationship of associated objects and indicates that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Here, A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0046] In this application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single item(s) or plural item(s). For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0047] It should be understood that in various embodiments of this application, the magnitudes of the serial numbers of the above - mentioned processes do not imply the sequence of execution. Some or all of the steps can be executed in parallel or successively. The execution sequence of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0048] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The singular forms of "a", "the", and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0049] The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from regular biochemical reagent stores unless otherwise specified.

[0050] The first aspect of the embodiments of this application provides a strain based on flagellin and LPS - attenuated Salmonella typhimurium EN - VNP strain, which is formed by sequentially knocking out or silencing the flagellin genes fliC, fljB, phosphoethanolamine transferase gene eptA, 4 - amino - 4 - deoxy - L - arabinose transferase gene arnT, phosphotransferase yeiU and knocking in or overexpressing the phosphotransferase gene lpxE on the basis of Salmonella VNP 20009 strain.

[0051] The Salmonella VNP 20009 strain used in the present invention is derived from the ATCC strain preservation center (ATCC202165).

[0052] In some embodiments, the nucleotide sequence of the gene fliC is as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is as shown in SEQ ID NO.2; the nucleotide sequence of the gene fljB is as shown in SEQ ID NO.3, and the nucleotide sequence of the encoded protein is as shown in SEQ ID NO.4; the nucleotide sequence of the phosphoethanolamine transferase gene eptA is as shown in SEQ ID NO.5, and the nucleotide sequence of the encoded protein is as shown in SEQ ID NO.6; the nucleotide sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is as shown in SEQ ID NO.7, and the nucleotide sequence of the encoded protein is as shown in SEQ ID NO.8; the nucleotide sequence of the phosphotransferase gene yeiU is as shown in SEQ ID NO.9, and the nucleotide sequence of the encoded protein is as shown in SEQ ID NO.10; the nucleotide sequence of the phosphotransferase gene lpxE is as shown in SEQ ID NO.11, and the nucleotide sequence of the encoded protein is as shown in SEQ ID NO.12.

[0053] In the second aspect of the embodiments of the present application, a preparation method of an attenuated Salmonella typhimurium EN-VNP strain based on flagellin and LPS is provided, including the following steps:

[0054] (1) Construct a ΔfliC strain, knock out the fliC gene through the CRISPR / Cas9 gene editing system, and use the designed sgRNA sequence and homologous arm sequence for gene knockout;

[0055] (2) On the basis of the ΔfliC strain, construct a ΔfljB strain, knock out the fljB gene through the CRISPR / Cas9 system, and use the designed sgRNA sequence and homologous arm sequence for gene knockout;

[0056] (3) On the basis of the ΔfliCΔfljB strain, knock out the eptA gene, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout;

[0057] (4) On the basis of the ΔfliCΔfljBΔeptA strain, knock out the arnT gene, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout;

[0058] (5) On the basis of the ΔfliCΔfljBΔeptAΔarnT strain, knock out the yeiU gene, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout;

[0059] (6) Overexpress the lpxE gene in the ΔfliCΔfljBΔeptAΔarnTΔyeiU strain, and use a plasmid containing the J23100 constitutive promoter and Kan+ resistance as the expression vector for the lpxE gene for gene overexpression.

[0060] In some embodiments, in step (1), the sgRNA sequence targets 5'-AACGAAATCGACCGTGTATC-3', the nucleotide sequence of the upstream homologous arm is as shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm is as shown in SEQ ID NO.14;

[0061] In step (2), the sgRNA sequence targets 5'-GTTTACGGTATTGCCCAGGT-3', the nucleotide sequence of the upstream homologous arm is as shown in SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm is as shown in SEQ ID NO.16;

[0062] In step (3), the sgRNA sequence targets 5'-GGCGAATCATTGGGTGAAAA-3', the nucleotide sequence of the upstream homologous arm is as shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm is as shown in SEQ ID NO.18;

[0063] In step (4), the sgRNA sequence targets 5'-TCAACAGCCGCCTGCTCTGG-3', the nucleotide sequence of the upstream homologous arm is as shown in SEQ ID NO.19, and the nucleotide sequence of the downstream homologous arm is as shown in SEQ ID NO.20;

[0064] In step (5), the sgRNA sequence targets 5'-TGCGCAGCGAGTTTAACGGT-3', the nucleotide sequence of the upstream homologous arm is as shown in SEQ ID NO.21, and the nucleotide sequence of the downstream homologous arm is as shown in SEQ ID NO.22;

[0065] In step (6), use a plasmid containing the J23100 constitutive promoter and Kan+ resistance as the expression vector J23-lpxE for the lpxE gene.

[0066] In some embodiments, the culture conditions of the strain are 37°C until OD600 = 0.6 - 0.8; the electrotransformation conditions are set to 2400V, 200Ω; use arabinose to induce the expression of Cas9 protein at a concentration of 3mg / mL and an incubation time of 1 hour; remove the pTarget plasmid by IPTG induction, culture overnight and passage 2 times to lose Amp resistance.

[0067] In the third aspect of the embodiments of the present application, a method using the attenuated Salmonella enterica serovar Typhimurium EN-VNP strain as a delivery vector is provided for delivering a therapeutic protein or drug to tumor cells.

[0068] In the fourth aspect of the embodiments of the present application, a method for detecting the knockout or silencing effect of specific genes in the flagellin- and LPS-attenuated Salmonella enterica serovar Typhimurium EN-VNP strain is provided, using PCR and electrophoresis techniques, and the sequences shown in SEQ ID NO.25 to SEQ ID NO.28 for identification.

[0069] The application of the flagellin- and LPS-attenuated Salmonella enterica serovar Typhimurium EN-VNP strain provided in the fifth aspect of the embodiments of the present application can activate the immune system.

[0070] The application of the flagellin- and LPS-attenuated Salmonella enterica serovar Typhimurium EN-VNP strain provided in the sixth aspect of the embodiments of the present application in the preparation of a vaccine preparation activates the immune system and exerts the immune activation effect of the vaccine.

[0071] The application of the flagellin- and LPS-attenuated Salmonella enterica serovar Typhimurium EN-VNP strain provided in the seventh aspect of the embodiments of the present application in the preparation of an anti-tumor drug activates the immune system and exerts the immune activation effect of activating anti-tumor immunity.

[0072] The Salmonella enterica serovar Typhimurium VNP 20009 used in the embodiments of the present invention is derived from the ATCC strain preservation center (ATCC202165).

[0073] Example 1

[0074] Construction of attenuated Salmonella enterica serovar Typhimurium EN-VNP

[0075] 1. Construction of the ΔfliC strain

[0076] 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 the Cas9 protein, and the pTarget plasmid has Amp resistance and carries the sgRNA sequence and homologous recombination repair sequence. The sequence of the gene fliC is shown in SEQ ID NO.1, and the sequence of the encoded protein is shown in SEQ ID NO.2.

[0077] The designed 20bp sgRNA sequence for targeting is: 5’-AACGAAATCGACCGTGTATC-3’. The nucleotide sequence of the upstream homologous arm for knocking out the gene fliC is shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.14.

[0078] Preparation of VNP containing CRISPR / Cas9 plasmid: Inoculate VNP monoclonal into 2 mL of LB medium and culture at 37 °C until OD600 = 0.6. Centrifuge to collect the bacteria, wash the bacteria 3 times with pre-cooled 10% glycerol, and then resuspend with 100 μL of 10% glycerol. Add 1 μg of Cas9 plasmid to the competent cells and perform electroporation with the conditions set as: 2400 V, 200 Ω. Then spread on the plate and culture at 30 °C with Kan resistance overnight. Pick the monoclonal, which is VNP-cas9.

[0079] Preparation of VNP-cas9 strain containing pTarget plasmid: Inoculate VNP-cas9 monoclonal into 2 ml of LB medium and culture at 30 °C until OD600 = 0.1 - 0.2. Add arabinose for induction at a concentration of 3 mg / mL and incubate for 1 hr. Centrifuge to collect the bacteria, wash 3 times with 10% glycerol, and then resuspend with 100 μL of 10% glycerol. Add 1 μg of pTarget plasmid to the competent cells and perform electroporation. Then spread on the LB plate with Kan+ and Amp+ and incubate for more than 16 hr. Pick the monoclonal and perform amplification culture.

[0080] Removal of single plasmid: Add IPTG to the medium and culture overnight, passage 2 generations to remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick the monoclonal and culture it for passage and preservation of the seeds, which is VNPΔfliC.

[0081] Example 2

[0082] Construction of ΔfljB strain based on VNPΔfliC

[0083] The sequence of gene fljB is shown as SEQ ID NO.3, and the sequence of the encoded protein is shown as SEQ ID NO.4.

[0084] The designed 20bp sgRNA sequence for targeting is: 5’-GTTTACGGTATTGCCCAGGT-3’. The nucleotide sequence of the upstream homologous arm for knocking out gene fljB is shown as SEQ ID NO.15, and the nucleotide sequence of the downstream homologous arm is shown as SEQ ID NO.16.

[0085] Preparation of VNPΔfliC strain containing pTarget plasmid: Inoculate VNPΔfliC monoclonal (containing cas9 plasmid, Kan resistant) into 2 mL of LB medium, culture at 30 °C until OD600 = 0.1 - 0.2, add arabinose for induction at a concentration of 3 mg / mL, incubate for 1 hr, centrifuge to collect the bacteria, wash 3 times with 10% glycerol, and then resuspend with 100 μL of 10% glycerol. Add 1 μg of pTarget plasmid to the competent cells for electroporation. Then spread on an LB plate, with Kan+ and Amp+, incubate for more than 16 hr, pick monoclonal colonies, and amplify and culture them.

[0086] Removal of single plasmid: Add IPTG to the medium, culture overnight, passage 2 generations to remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick monoclonal colonies, culture them by passage, and preserve the seeds, which is VNPΔfliCΔfljB.

[0087] 3. Knock out the eptA gene based on VNPΔfliCΔfljB

[0088] The sequence of the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.5, and the sequence of the encoded protein is shown in SEQ ID NO.6.

[0089] Design a 20bp sgRNA sequence for targeting: 5’-GGCGAATCATTGGGTGAAAA-3’. The nucleotide sequence of the upstream homologous arm for knocking out the phosphoethanolamine transferase gene eptA is shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.18.

[0090] Preparation of VNPΔfliCΔfljB strain containing pTarget plasmid: Inoculate VNPΔfliCΔfljB monoclonal (containing cas9 plasmid, Kan resistant) into 2 ml of LB medium, culture at 30 °C until OD600 = 0.1 - 0.2, add arabinose for induction at a concentration of 3 mg / mL, incubate for 1 hr, centrifuge to collect the bacteria, wash 3 times with 10% glycerol, and then resuspend with 100 μL of 10% glycerol. Add 1 μg of pTarget plasmid to the competent cells for electroporation. Then spread on an LB plate, with Kan+ and Amp+, incubate for more than 16 hr, pick monoclonal colonies, and amplify and culture them.

[0091] Removal of single plasmid: Add IPTG to the medium, culture overnight, passage 2 generations to remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick monoclonal colonies, culture them by passage, and preserve the seeds, which is VNPΔfliCΔfljBΔeptA.

[0092] 4. Knock out the arnT gene based on VNPΔfliCΔfljBΔeptA

[0093] The sequence of the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.7, and the sequence of the encoded protein is shown in SEQ ID NO.8.

[0094] Design a 20bp sgRNA sequence for targeting: 5’-TCAACAGCCGCCTGCTCTGG-3’. The nucleotide sequence of the upstream homologous arm for knocking out the 4-amino-4-deoxy-L-arabinose transferase gene arnT is shown in SEQ ID NO.19, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.20.

[0095] Preparation of VNPΔfliCΔfljBΔeptA strain containing pTarget plasmid: Inoculate a VNPΔfliCΔfljBΔeptA monoclonal (containing cas9 plasmid, Kan resistant) into 2 mL of LB medium, culture at 30 °C until OD600 = 0.1 - 0.2, add arabinose for induction at a concentration of 3 mg / mL, incubate for 1 hr, centrifuge to collect the bacteria, wash 3 times with 10% glycerol, and then resuspend with 100 μL of 10% glycerol. Add 1 μg of pTarget plasmid to the competent cells and perform electroporation. Then spread on an LB plate, Kan+ and Amp+, incubate for more than 16 hr, pick monoclonal colonies, and perform amplification culture.

[0096] Remove the single plasmid: Add IPTG to the medium, culture overnight, passage for 2 generations to remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick monoclonal colonies, culture and passage to preserve the seeds, which is VNPΔfliCΔfljBΔeptAΔarnT.

[0097] 5. Knock out the yeiU gene based on VNPΔFliCΔFljBΔeptAΔarnT

[0098] The sequence of the phosphotransferase gene yeiU is shown in SEQ ID NO.9, and the sequence of the encoded protein is shown in SEQ ID NO.10.

[0099] Design a 20bp sgRNA sequence for targeting: 5’-TGCGCAGCGAGTTTAACGGT-3’. The nucleotide sequence of the upstream homologous arm for knocking out the phosphotransferase gene yeiU is shown in SEQ ID NO.21, and the nucleotide sequence of the downstream homologous arm is shown in SEQ ID NO.22.

[0100] Preparation of VNPΔFliCΔFljBΔeptAΔarnT strain containing pTarget plasmid: Inoculate a VNPΔFliCΔFljBΔeptAΔarnT monoclonal (containing cas9 plasmid, Kan resistant) into 2 mL of LB medium and culture at 30 °C until OD600 = 0.1 - 0.2. Add arabinose for induction at a concentration of 3 mg / mL, incubate for 1 hr, collect the bacteria by centrifugation, wash 3 times with 10% glycerol, and then resuspend with 100 μL of 10% glycerol. Add 1 μg of pTarget plasmid into the competent cells and perform electroporation. Then spread on an LB plate with Kan+ and Amp+, incubate for more than 16 hr, pick monoclonal colonies, and amplify and culture them.

[0101] Removal of single plasmid: Add IPTG into the medium, culture overnight, passage 2 generations to remove the pTarget plasmid. At this time, VNP loses Amp resistance. Pick monoclonal colonies, culture and passage them to preserve the seeds, which is VNPΔfliCΔfljBΔeptAΔarnTΔyeiU.

[0102] 6. Overexpress the lpxE gene based on VNPΔfliCΔfljBΔeptAΔarnTΔyeiU

[0103] The sequence of the phosphotransferase gene lpxE is shown in SEQ ID NO.11, and the sequence of the encoded protein is shown in SEQ ID NO.12.

[0104] Use a plasmid containing the J23100 constitutive promoter and Kan+ resistance as the expression vector for the lpxE gene (J23-lpxE).

[0105] Preparation of VNPΔfliCΔfljBΔeptAΔarnTΔyeiU strain: Inoculate a VNPΔfliCΔfljBΔeptAΔarnTΔyeiU monoclonal (without resistance) into 2 mL of LB medium and culture at 37 °C until OD600 = 0.6 - 0.8. Collect the bacteria by centrifugation, wash 3 times with 10% glycerol, and then resuspend with 100 μL of 10% glycerol. Add 1 μg of J23-lpxE plasmid into the competent cells and perform electroporation. Then spread on a Kan+ LB plate, incubate for more than 16 hr, pick monoclonal colonies, and amplify and culture them.

[0106] The obtained strain after sequentially knocking out the flagellin gene fliC, fljB, phosphoethanolamine transferase gene eptA, 4-amino-4-deoxy-L-arabinose transferase gene arnT, phosphotransferase yeiU, and knocking in or overexpressing the phosphotransferase gene lpxE is named the EN-VNP strain.

[0107] 7. Identify the knockout situation of the eptA gene with the following primers:

[0108] Forward primer: 5’-tgccttgagcatcaaccgtg-3’

[0109] Reverse primer: 5’-tcggcgtgctgatgactatc-3’

[0110] The sequence interval of the wild strain by PCR sequencing is: 4440726 - 4443344, theoretically 2618 nt in total; the actual sequencing result of the mutant strain is 974 nt, and the sequence is shown as SEQ ID NO.25.

[0111] 8. To identify the knockout situation of the arnT gene with the following primers:

[0112] Forward primer: 5’-atattgcgcgcggcataacg-3’

[0113] Reverse primer: 5’-agactcagtagcgcataggc-3’

[0114] The sequence interval of the wild strain by PCR sequencing is: 2350620 - 2353357, theoretically 2737 nt in total; the actual sequencing result of the mutant strain is 1092 nt, and the sequence is shown as SEQ ID NO.26.

[0115] 9. To identify the knockout situation of the yeiU gene with the following primers:

[0116] Forward primer: 5’-gatcgtcaactgattcatgc-3’

[0117] Reverse primer: 5’-tcggttgagatttgaccatg-3’

[0118] The sequence interval of the wild strain by PCR sequencing is: 2254197 - 2256563, theoretically 1646 nt in total; the actual sequencing result of the mutant strain is 926 nt, and the sequence is shown as SEQ ID NO.27.

[0119] 10. To identify the transfer situation of the lpxE gene with the following primers:

[0120] Forward primer: 5’-agtctggaaagaaatgcat-3’

[0121] Reverse primer: 5’-cgcaatacacataatcctgg-3’

[0122] The theoretical total by PCR sequencing is 2612 nt; the actual sequencing result of the mutant strain is 1991 nt, and the sequence is shown as SEQ ID NO.28.

[0123] The PCR electrophoresis identification diagram is as shown in Figure 3 , indicating that eptA, arnT, and yeiU have been successfully knocked out and lpxE has been successfully knocked in. Through electron microscopy observation, as shown in Figure 4 , flagella can be seen in the electron microscopy of the wild-type VNP 20009 ( Figure 4 ). For the strain EN-VNP (VNP VNPΔfliCΔfljBΔeptAΔarnTΔyeiU:lpxE) obtained after gene knockout, the knocked-out flagella can be seen in the electron microscopy photo ( Figure 5 ).

[0124] Example 3

[0125] A method of the present invention uses the attenuated Salmonella enterica serovar Typhimurium EN-VNP strain as a delivery vector to deliver therapeutic proteins or drugs to tumor cells.

[0126] Example 4

[0127] A method of the present invention for detecting the knockout or silencing effect of specific genes in the flagellin- and LPS-attenuated Salmonella enterica serovar Typhimurium EN-VNP strain uses PCR and electrophoresis techniques and the sequences shown in SEQ ID NO.25 to SEQ ID NO.28 for identification.

[0128] Example 5

[0129] An application of the flagellin- and LPS-attenuated Salmonella enterica serovar Typhimurium EN-VNP strain in the preparation of vaccine preparations. Given that EN-VNP has the same normal growth performance as VNP, the cell size is consistent with VNP and there is no significant change, so it has the same application as VNP.

[0130] Activate the carboxyl group of the surface protein of the EN-VNP strain with EDAC, bind it to the —NH2 group of amine-PEG3-biotin, and wrap biotin on the surface of the EN-VNP strain; then mix the biotinylated EN-VNP strain with the target protein (for example, RFP) and incubate to obtain a product with the antigen conjugated on the surface of the EN-VNP strain. Immunize mice with the EN-VNP strain with RFP antigen conjugated on the strain membrane surface on the 1st and 14th days, collect mouse serum on the 28th day, and detect the concentration of RFP-specific antibodies in the serum by ELISA. The concentration of antibodies induced against the RFP antigen conjugated on the bacterial surface is about 8 times that of the antibodies induced by the RFP antigen protein alone (1.6045 ± 0.269354 vs 0.197 ± 0.070268; the PBS group is: 0.059 ± 0.00743).

[0131] Example 6

[0132] Given that anti-tumor activity is a well-known and widely studied basic property of VNP, and given that EN-VNP has the same normal growth performance as VNP, with the cell size being consistent with that of VNP and showing no significant change, it thus has the same applications as VNP. An application of a flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain based on the present invention in the preparation of anti-tumor drugs.

[0133] Test Example 1

[0134] Inflammatory factor concentration detection test

[0135] Inoculate the EN-VNP monoclonal antibody prepared in Example 1 into 2 mL of LB medium and culture it at 37 °C until OD600 = 0.6 - 0.8. Centrifuge to collect the bacterial cells, wash them 3 times with PBS, and then resuspend them in 1 mL of PBS. Inject 1x106 into the abdominal cavity of 6 - 8-week-old BALB / C mice. The control groups are the PBS solution and the VNP2009 strain. After 5 days, detect the expression level of inflammatory factors in the mouse serum by ELISA. The results are as Figure 6 shown. The generation levels of inflammatory factors (such as IL-6, TNF-α) induced by EN-VNP decreased significantly and were almost equivalent to those of PBS; they were much lower than those of the VNP strain itself, so it has good safety.

[0136] 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 by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification, and their equivalents.

Claims

1. An attenuated Salmonella typhimurium EN-VNP strain based on flagellin and LPS, characterized in that: A strain formed by successively knocking out or silencing the flagellin genes fliC, fljB, phosphoethanolamine transferase gene eptA, 4-amino-4-deoxy-L-arabinose transferase gene arnT, phosphotransferase yeiU on the basis of Salmonella strain VNP 20009, and knocking in or overexpressing the phosphotransferase gene lpxE.

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

12.

3. The preparation method of the attenuated Salmonella typhimurium EN-VNP strain based on flagellin and LPS according to claim 1, characterized in that Comprising the following steps: (1) Construct a ΔfliC strain, knock out the fliC gene through the CRISPR / Cas9 gene editing system, and use the designed sgRNA sequence and homologous arm sequence for gene knockout; (2) On the basis of the ΔfliC strain, construct a ΔfljB strain, knock out the fljB gene through the CRISPR / Cas9 system, and use the designed sgRNA sequence and homologous arm sequence for gene knockout; (3) Knock out the eptA gene on the basis of the ΔfliCΔfljB strain, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout; (4) Knock out the arnT gene on the basis of the ΔfliCΔfljBΔeptA strain, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout; (5) Knock out the yeiU gene on the basis of the ΔfliCΔfljBΔeptAΔarnT strain, and use the designed sgRNA sequence and homologous arm sequence through the CRISPR / Cas9 system for gene knockout; (6) Overexpress the lpxE gene on the basis of the ΔfliCΔfljBΔeptAΔarnTΔyeiU strain, and use the plasmid containing the J23100 constitutive promoter and Kan+ resistance as the expression vector of the lpxE gene for gene overexpression.

4. The preparation method according to claim 3, wherein: In step (1), the sgRNA sequence targets 5’-AACGAAATCGACCGTGTATC-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.13, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.14; In step (2), the sgRNA sequence targets 5’-GTTTACGGTATTGCCCAGGT-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.15, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.16; In step (3), the sgRNA sequence targets 5’-GGCGAATCATTGGGTGAAAA-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.17, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.18; In step (4), the sgRNA sequence targets 5’-TCAACAGCCGCCTGCTCTGG-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.19, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.20; In step (5), the sgRNA sequence targets 5’-TGCGCAGCGAGTTTAACGGT-3’, the nucleotide sequence of the upstream homology arm is as shown in SEQ ID NO.21, and the nucleotide sequence of the downstream homology arm is as shown in SEQ ID NO.22; In step (6), a plasmid containing the J23100 constitutive promoter and Kan+ resistance is used as the expression vector J23-lpxE for the lpxE gene.

5. The preparation method according to claim 3, wherein: The culture conditions for the strain are 37 °C until OD600 = 0.6 - 0.8; the electrotransformation conditions are set at 2400 V, 200 Ω; the expression of the Cas9 protein is induced using arabinose at a concentration of 3 mg / mL, and the incubation time is 1 hour; the pTarget plasmid is removed by IPTG induction, and the cells are cultured overnight and passaged twice to lose Amp resistance.

6. A method using the flagellin and LPS attenuated Salmonella typhimurium EN-VNP strain described in claim 1 as a delivery vector, characterized in that: For delivering a therapeutic protein or drug to tumor cells.

7. A method for detecting the knockout or silencing effect of a specific gene in the flagellin and LPS attenuated Salmonella typhimurium EN-VNP strain according to claim 1, characterized in that: Identification is carried out using PCR and electrophoresis techniques, as well as the sequences shown in SEQ ID NO.25 to SEQ ID NO.

28.

8. The application of the flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain according to claim 1, characterized in that: Capable of activating immunity.

9. Use of the flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain according to claim 8 in the preparation of a vaccine preparation, characterized in that: By activating immunity, exerting the immune activation effect of the vaccine.

10. Use of the flagellin- and LPS-attenuated Salmonella typhimurium EN-VNP strain according to claim 8 in the preparation of an anti-tumor drug, characterized in that: By activating immunity, exerting the immune activation effect of activating anti-tumor immunity.

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