Eukaryotic expression vector, bacterium containing same, and use

Abstract

The present application relates to the field of genetic engineering and biological therapy, and provides a eukaryotic expression vector, a gram-negative bacterium containing the eukaryotic expression vector, and a use of the eukaryotic expression vector in the preparation of antitumor drugs and the treatment of tumors. By selecting and combining a phage-derived promoter, a specific phage RNA polymerase gene, a membrane-disrupting protein gene and a eukaryotic expression drug protein gene, the eukaryotic expression vector is divided into three modules: (1) a phage-derived RNA polymerase gene transcription module; (2) a membrane-disrupting protein gene expression module; and (3) a eukaryotic drug protein gene expression module. By means of organic combination of the three modules, a eukaryotic drug delivery system is constructed, and a system and method for stable and efficient eukaryotic drug protein expression, drug delivery and drug killing are implemented.

Description

A eukaryotic expression vector, bacteria containing the same and applications thereof

[0001] This application claims priority to a prior application, patent application number 202410136658.9, entitled “A Eukaryotic Expression Vector, Bacteria Containing the Same, and Applications thereof,” filed with the State Intellectual Property Office of China on January 31, 2024. The entire text of that prior application is incorporated herein by reference. Technical Field

[0002] The present invention relates to a eukaryotic expression vector, bacteria containing the same and applications, and in particular to a constitutive eukaryotic drug mRNA / DNA mixed dual delivery cytoplasmic expression system containing a dual phage-derived promoter, bacteria containing the same and applications, belonging to the fields of genetic engineering and biotherapy. Background Art

[0003] Cancer is one of the leading causes of death worldwide, and its prevalence is increasing every year. Solid tumors account for approximately 90% of all malignant tumors, such as sarcomas, melanomas, breast cancer, lung cancer, colon cancer, and prostate cancer.

[0004] The tumor microenvironment of solid tumors shares common characteristics, including abnormal tumor vasculature, excessive connective tissue, immunosuppression, an acidic environment, and hypoxic regions. Furthermore, this abnormal microenvironment presents a natural barrier that is difficult for traditional therapeutic drugs to penetrate, making it difficult for chemotherapy drugs and antibodies to diffuse within the solid tumor microenvironment. Furthermore, a lack of oxygen free radicals contributes to tumor resistance to chemotherapy and radiotherapy.

[0005] Due to the hypoxic environment within solid tumors, facultative and obligate anaerobes are able to invade tumors and inhibit their growth and therefore can be used as therapeutic agents or carriers with great potential.

[0006] As early as 1868, German physician W. Busch first reported that in some cancer patients, bacterial infection (Streptococcus pneumoniae) inhibited tumor growth, or even completely eliminated it. Within the next three decades, American physician Coley and German physician Fehleisen independently reported that bacterial infection could inhibit tumors. These early studies were controversial due to difficulties in replicating the results and controlling the virulence of the bacteria. However, rigorous animal experiments later demonstrated that bacterial infection could indeed reduce tumor size and activate the host immune system during treatment. In 1975, Carswell first reported that lipopolysaccharides produced by Gram-negative bacteria could stimulate the immune system to release tumor necrosis factor (TNF-α), which could lead to tumor cell death. Furthermore, some bacterial vaccines have also been shown to stimulate the immune system and thus treat tumors. Among them, Bacillus Calmette-Guérin (BCG) was the first biological agent used in clinical cancer treatment. BCG is a live attenuated bacterial suspension derived from the highly pathogenic bovine tuberculosis bacillus, which has been serially inoculated for 230 generations. It is used for the prevention of tuberculosis. A large number of experiments and clinical practices have confirmed that BCG is one of the most effective means of treating bladder cancer.

[0007] In recent years, with the rapid development of molecular biology and genetic engineering technologies, studies have revealed that some facultative or obligate anaerobes, such as the obligate anaerobic bacterium Clostridium and the probiotic Bifidobacterium, can target, colonize, and proliferate within solid tumors, potentially inducing tumor regression. Among these, the Gram-negative facultative anaerobic bacterium Salmonella enterica has the greatest potential for application.

[0008] Studies have shown that after Salmonella typhimurium is attenuated through different methods, its colonization ability in tumor tissue can reach 1000-10000 times that of normal tissue, and it can be used as a potential targeted drug for the treatment of tumors.

[0009] Regarding improvements in targeting tumors with recombinant bacteria, existing literature reports suggest that these bacteria can carry various types of drugs for targeted delivery to tumors. However, these results have been less than ideal. This may be due to a lack of comprehensive consideration of the bacterial efficiency of drug synthesis and delivery within tumors.

[0010] In addition, since bacteria are used as carriers for drug delivery, the drugs carried are limited by the bacteria's own conditions. First, bacteria are prokaryotes, and the proteins directly produced by their expression systems lack glycosylation modifications and chaperone proteins necessary for protein folding. Therefore, if the drug carried is a eukaryotic protein, it is difficult to express a correctly folded or glycosylated protein, thereby affecting its efficacy. Secondly, if the expressed drug protein itself is toxic to bacteria, the bacteria will not be able to express it efficiently.

[0011] Therefore, when using bacteria as a vector to carry eukaryotically expressed drug genes for expression in eukaryotic cells, since the DNA transcription process in eukaryotic systems occurs in the cell nucleus, direct bacterial delivery of DNA requires the DNA to be shuttled into the cell nucleus. However, the probability of exogenous DNA entering the cell nucleus is very low. Only during mitosis does the nuclear membrane disaggregate, allowing exogenous DNA to enter the nuclear region. Therefore, adopting a cytoplasmic expression model is key to increasing presentation efficiency.

[0012] The 2023 Nobel Prize in Physiology or Medicine was awarded to scientists Katalin Karikó and Drew Weissman for their contributions to nucleobase modification. Their contributions enabled the development of effective messenger RNA (mRNA) vaccines against COVID-19. The success of mRNA vaccines over DNA vaccines suggests that direct cytoplasmic expression of mRNA offers significant advantages over DNA expression, which requires entry into the cell nucleus.

[0013] U.S. Patent No. 10987432B2 reports a bacterial vector-based cytoplasmic expression method. This method involves using Salmonella to deliver a hybrid DNA / mRNA vector carrying a drug gene controlled by both a CMV promoter and a T7 promoter. Simultaneously, an autogene consisting of a T7 promoter and a T7 RNA polymerase gene is located downstream to automatically enhance gene expression. The goal is to continuously synthesize T7 RNA polymerase in the cytoplasm through the T7 RNA polymerase gene carried by the bacteria, forming a positive feedback loop and simultaneously synthesizing the target drug gene.

[0014] However, this design is not a true cytoplasmic expression vector. In this design, the CMV promoter (which initiates transcription in the nucleus) and the polyA signaling regulatory element (which is required for mRNA maturation in the nucleus) rather than polyadenylation are not able to effectively form a polyA mRNA tail in the cytoplasm. Therefore, this is not a specific and efficient cytoplasmic expression method, but rather a nuclear import-dependent cytoplasmic expression model.

[0015] Furthermore, the two T7 promoters in the drug gene vector regulate the transcription of the drug gene and T7 RNA polymerase, respectively. This design may lead to post-transcriptional mRNA diversity, ultimately reducing the number of effective mRNAs presented to the cytoplasm. Furthermore, the expression pattern of the membrane-permeabilizing protein has not been optimized, which may affect its ultimate presentation efficiency and thus the overall efficacy.

[0016] In summary, there is an urgent need for a eukaryotic drug protein system and method that can stably and efficiently express and release bacteria in tumors in vivo. Summary of the Invention

[0017] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terminology and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the relevant fields and are standard procedures.

[0018] In response to the above-mentioned defects in the prior art, the present invention establishes a constitutive eukaryotic drug mRNA / DNA mixed dual delivery cytoplasmic expression system containing dual phage-derived promoters.

[0019] To this end, the present invention provides a constitutive eukaryotic drug mRNA / DNA hybrid dual delivery cytoplasmic expression system containing dual phage-derived promoters, comprising three modules: (1) a phage-derived promoter and RNA polymerase transcription control module; (2) a delivery module; and (3) a eukaryotic drug mRNA transcription module and a phage-derived RNA polymerase self-enhancing module. By organically combining these three modules and taking advantage of the characteristics of bacteria, the problem of bacterial vectors being difficult to carry eukaryotic drugs is resolved.

[0020] 1. Phage-derived promoter and RNA polymerase transcription control module

[0021] In order to achieve stable and efficient gene expression in the tumor microenvironment, it is necessary to use a high-intensity promoter expression system that is not affected by the environment. The present invention adopts the constitutive artificial promoter lacUV5 to control the expression of the phage-derived T7 RNA polymerase gene, and integrates this expression cassette into the chromosome of the bacteria to achieve stable inheritance. Because the T7 RNA polymerase specifically recognizes the T7 promoter, it is possible to achieve stable and high-intensity expression of the gene controlled by the T7 promoter. Therefore, the delivery module and the eukaryotic drug mRNA transcription module and the phage-derived RNA polymerase self-enhancing module are respectively placed behind the T7 promoter and placed on the plasmid for expression regulation.

[0022] 2. Delivery Module

[0023] When bacteria invade tumor cells, phagocytic vesicles are formed. The presence of phagocytic vesicles will limit the further release of bacteria and the drugs they synthesize into the cells. In order to solve this problem, the present invention uses listeriolysin-O (LLO) as a membrane-breaking protein to achieve the purpose of drug delivery. In order to increase its efficiency as a membrane-breaking protein, the present invention places the LLO gene on an expression vector plasmid and uses a T7 promoter to control expression. Not only does it increase the overall expression level of LLO, but by expressing it synchronously with the mRNA of eukaryotic drugs in advance, it can efficiently break the membrane while forming phagocytic vesicles, which is compared with patent US10987432B2 and improves the overall presentation efficiency.

[0024] 3. Eukaryotic drug mRNA transcription module and phage-derived RNA polymerase self-enhancing module

[0025] (1) Selection of eukaryotic drugs

[0026] In order to achieve a precise drug that exerts an efficient killing effect only in tumor cells, the present invention uses the N-terminal fragment of the eukaryotic cell pyroptosis protein. Pyroptosis is a form of cell death that is crucial for immunity. It is usually induced by the typical caspase-1 inflammasome or activated caspase-4, -5 and -11. Activated caspases cleave the pyroptosis protein gasdermin (GSDM) in the intermediate linker of the pyroptosis protein gasdermin (including GSDMA, GSDMB, GSDMC, GSDMD, GSDME) to release its gasdermin-N-terminal fragment domain, which is in an activated state. The fragment can penetrate the cell membrane, thereby inducing cell pyroptosis. Studies have found that after caspase activation, GSDMD is proteolytically cleaved at the Asp 275 site, while GSDME is specifically cleaved at the Asp 270 site by caspase-3 and broken into two parts and becomes active. The N-terminal protein of the broken GSDMD or GSDME protein has pore-punching activity and can insert into the cell membrane to form holes, thereby inducing cell pyroptosis. GSDME is silenced in most cancer cells but expressed in many normal tissues. Human primary cells exhibit GSDME-dependent pyroptosis after activation of caspase-3 by chemotherapeutic drugs. The C-terminus of GSDMD or GSDME acts as an inhibitor, inhibiting the activity of the N-terminus in the uncleaved state. The N-terminal cleavage product of GSDM localizes to the plasma membrane by anchoring to membrane lipids and interacts specifically with phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] in the inner layer of mammalian cell membranes, through the negatively charged head groups of [PI(4)P] and [PI(4,5)P] and the positively charged surface of GSDM-N exposed after cleavage. Lipid binding allows GSDM-N to insert into the lipid bilayer and induce membrane cohesion, forming extensive pores with an inner diameter of 10-14 nm. Osmotic pressure is disrupted by pore formation, leading to cell swelling and lysis, which are the morphological hallmarks of pyroptosis. These extensive pores also serve as protein secretion channels, promoting the secretion of inflammatory cytokines, thereby achieving a rapid innate immune response. Due to the lack of [PI(4)P] and [PI(4,5)P] outside the cell, GSDM-N leaked outside the cell is non-toxic. Since GSDM-N has an indiscriminate effect on the cell's inner membrane, it is also toxic to bacteria.

[0027] Two key characteristics of GSDM-N: its eukaryotic origin, the need for glycosylation, and its indiscriminate attack on the bacterial inner membrane prevent direct bacterial expression of the GSDM-N gene. Therefore, the strategy of using a eukaryotic delivery system rather than direct bacterial expression is a promising one.

[0028] (2) Selection of eukaryotic mRNA cytoplasmic expression system

[0029] Studies have shown that Salmonella can carry eukaryotic plasmids and can present eukaryotic plasmids into cells. However, since plasmid DNA needs to enter the cell nucleus to be transcribed into mRNA and then start translation, this efficiency is very low. In order to solve this problem, the present invention uses a bacterial system to directly synthesize eukaryotic mRNA in bacteria, and the mRNA / DNA vector can be directly delivered to the cytoplasm of cancer cells through the presentation system, and the eukaryotic drug protein is synthesized by utilizing the translation system of the cytoplasm. The structure of the mature mRNA includes the internal entry sequence (IRES) of the virus, the kozak sequence, the orf region of the eukaryotic drug, the 3'-UTR region, and the polyA tail. In the present invention, since the bacteria themselves lack the relevant enzymes for mRNA capping, the internal entry sequence IRES of the virus is used instead, which can successfully achieve direct synthesis of bacteria and carry the mRNA containing IRES to present it to the cytoplasm of cancer cells.

[0030] (3) Phage-derived RNA polymerase self-enhancing system based on mRNA / DNA mixed expression

[0031] Although bacteria can directly present the synthesized mRNA into the cytoplasm to achieve expression. However, due to the low efficiency of bacterial presentation of nucleic acids, it needs to be further improved. In addition to adjusting the spatiotemporal expression of LLO as described above, the method of the present invention is further increased by self-enhancement of phage-derived RNA polymerase. Compared with the method of patent US10987432B2, this patent completely abandons the mode of nuclear expression and two monocistronic (double T7 promoter controls self-enhanced transcription) and adopts a pure cytoplasmic expression method. The eukaryotic promoter CMV is removed, and the redundant design of the polyA signal (BGH polyA site "AATAAA") regulatory element (this element needs to participate in the maturation process of mRNA in the nucleus) is abandoned. Instead, it is directly designed into a single mRNA bicistronic transcription mode, and an IRES is directly connected after the single T7 promoter to control the stable expression of the drug gene and 3'-UTR, and a second IRES is added to control the stable expression of T7 RNA polymerase and 3'-UTR, and the polyA sequence is directly used as the 3' end of the mRNA downstream. This design can achieve an efficient mRNA / DNA mixed presentation mode.

[0032] During the tumor treatment process of the present invention, bacteria enter tumor tissue through the circulatory system and colonize there. Stable expression of T7 RNA polymerase within the bacteria promotes the massive synthesis of the GSDM-N fragment and bicistronic mRNA for T7 RNA polymerase located on the plasmid, as well as the stable and high expression of the drug delivery module, the membrane-breaking protein LLO. When the bacteria approach cancer cells in the tumor tissue, they use their own invasion system to induce endocytosis of the cancer cells, forming phagocytic vesicles containing the bacteria. The phagocytic vesicles then fuse with vacuoles such as lysosomes within the cell, thereby lowering the pH inside the phagocytic vesicles. At this time, the membrane-breaking protein LLO expressed by the bacteria is activated, causing the GSDM-N fragment, bicistronic mRNA for T7 RNA polymerase, and vector DNA to be released into the cytoplasm, where translation synthesis is initiated. The generated T7 RNA polymerase acts again on the vector DNA, initiating the next round of transcription. The accumulated GSDM-N fragments take effect, ultimately leading to pyroptosis of the cancer cells. This process is repeated continuously as the bacteria grow and replicate within the tumor, causing the rapid digestion of tumor cells. The characteristics of the present invention can only be achieved based on a eukaryotic drug mRNA / DNA mixed dual delivery cytoplasmic expression system.

[0033] In the present invention, a promoter containing two phage sources is used to control the self-enhanced transcription of the GSDM-N fragment and T7 RNA polymerase and the synchronous expression of the membrane-breaking protein LLO, respectively, and the corresponding phage RNA polymerase gene controlled by the constitutively expressed promoter lacUV5 is integrated into the host bacterial chromosome.

[0034] In a preferred embodiment of the present invention, the dual phage-derived promoter is selected from the group consisting of T7 (SEQ ID No. 1), T3 (SEQ ID No. 2) and SP6 promoter (SEQ ID No. 3).

[0035] In a more preferred embodiment of the present invention, the dual phage-derived promoter is the T7 promoter (SEQ ID No. 1).

[0036] In a preferred embodiment of the present invention, the specific phage RNA polymerase gene is a gene corresponding to a promoter derived from a biphage.

[0037] In a more preferred embodiment of the present invention, the specific phage RNA polymerase gene is selected from the T7 RNA polymerase gene (SEQ ID No.4) and T7 RNA polymerase protein sequence (SEQ ID No.5) after Salmonella codon optimization, the T3 RNA polymerase gene (SEQ ID No.6) and T3 RNA polymerase protein sequence (SEQ ID No.7) and the SP6 RNA polymerase gene (SEQ ID No.8) and SP6 RNA polymerase protein sequence (SEQ ID No.9).

[0038] In a further preferred embodiment of the present invention, the specific bacteriophage RNA polymerase gene is a T7 RNA polymerase gene.

[0039] In order to stably and efficiently express the drug protein, the present invention places the drug protein gene on a multi-copy expression plasmid vector and controls it through a T7 promoter to achieve maximum expression of the drug protein gene.

[0040] In order to achieve higher intensity expression, in a preferred embodiment of the present invention, a T7 phage RNA polymerase expression system is used, which includes T7 RNA polymerase and T7 promoter to control the expression of the target drug protein.

[0041] The T7 RNA polymerase system, derived from the expression system of the Escherichia coli T7 bacteriophage, is widely used in E. coli gene expression due to its robust protein expression capabilities. T7 RNA polymerase is characterized by its ability to specifically recognize the T7 promoter and is unaffected by environmental factors. Furthermore, its RNA synthesis rate is five times that of E. coli RNA polymerase, enabling high-intensity expression of target proteins. Furthermore, T7 RNA polymerase transcription is independent of other transcription factors, enabling independent transcription initiation even within the cytoplasm of eukaryotic cells. This offers significant advantages for cytoplasmic expression systems.

[0042] In order to achieve stable expression without being affected by bacterial internal metabolism and the tumor microenvironment, in a more preferred embodiment of the present invention, a constitutively expressed lacUV5 promoter (SEQ ID No. 10) is further used to control the expression of a specific phage RNA polymerase gene.

[0043] The lacUV5 promoter is very similar to the classic lac promoter. Compared to the lac promoter, it contains only a two-base-pair mutation in the -10 region. The lacUV5 promoter does not require an additional activator and can drive high levels of gene expression. Although it does not require an activator, expression from the lacUV5 promoter is regulated by the LacI repressor in E. coli and is inducible by IPTG. IPTG is an effective inducer when used in concentrations ranging from 100 μM to 1.5 mM.

[0044] Since Salmonella has lost the lacI gene and the entire lac operon during evolution, the lacUV5 promoter is an excellent constitutive expression system in Salmonella.

[0045] Therefore, in a more preferred embodiment of the present invention, a T7 RNA polymerase expression cassette controlled by a lacUV5 promoter is further transferred into the chromosome of Salmonella, thereby achieving stable constitutive expression.

[0046] In a preferred embodiment of the present invention, the T7 polymerase and T7 promoter can be replaced with T3 polymerase and T3 promoter. Similar to the T7 RNA polymerase system, the T3 RNA polymerase system is an RNA polymerase system derived from T3 bacteriophage that highly specifically recognizes the T3 promoter sequence. Alternatively, the T7 polymerase and T7 promoter can be replaced with SP6 polymerase and SP6 promoter. Similar to the T7 RNA polymerase system, SP6 polymerase is also an RNA polymerase system derived from SP6 bacteriophage that highly specifically recognizes the SP6 promoter sequence. T3 RNA polymerase or SP6 RNA polymerase and its corresponding promoter can functionally replace T7 RNA polymerase to specifically control the expression of downstream target genes.

[0047] In a preferred embodiment of the present invention, a T7 promoter bicistronic tandem eukaryotic expression of a drug protein and T7 RNA polymerase is used. There are two advantages: First, when inside the bacteria, T7 polymerase transcribes the drug protein and T7 RNA polymerase to produce a single bicistronic mRNA product. A single product is more conducive to controlling the ratio of drug protein and T7 RNA polymerase to achieve optimal presentation efficiency. Second, using the T7 promoter to directly control T7 RNA polymerase can easily produce a self-reinforcing cycle of positive feedback, resulting in depletion of bacterial internal resources and causing bacterial death. Although the T7 RNA polymerase used in the vector is a eukaryotic expression mode and lacks the necessary RBS, it can reduce the efficiency of prokaryotic expression in bacteria, but it cannot completely avoid leaky expression. Therefore, the self-reinforcing design of the bicistronic will increase the overall bacterial survival ability and efficiency.

[0048] In addition, in order to prevent the loss of the plasmid expression vector, an essential gene of Salmonella needs to be implanted during the construction of the vector, and the gene is removed from the chromosome of the host bacteria at the same time, thereby forming a balanced lethal control mechanism, that is, if the Salmonella loses the plasmid vector it carries, the bacteria will die quickly due to the lack of essential genes, and the surviving bacteria all carry the plasmid expression vector, thereby ensuring that the entire system stably and efficiently expresses protein drugs. The asd gene (SEQ ID No.11) of the genus Salmonella and the protein sequence (SEQ ID No.12) of asd encode aspartate beta-semialdehyde dehydrogenase (aspartate B-semialdehyde dehydrogenase), which is the enzyme required in the process of synthesizing diaminopimelate (DAP), an important component of the cell wall of Gram-negative bacteria. Knocking out the asd gene of Salmonella can cause the lysis and death of Salmonella, but when DAP is additionally supplemented in the culture medium or by causing Salmonella to carry a vector containing the asd gene, the normal growth of Salmonella can be maintained. Therefore, asd is used as an essential gene of the plasmid vector in the present invention. In a preferred embodiment of the present invention, the expression vector comprises the essential gene asd of Salmonella.

[0049] To achieve stable and efficient drug delivery, the present invention designs a precise drug delivery system. Its goal is to precisely release the drug protein synthesized by bacteria into tumor cells. This process is further divided into two steps: bacteria invading host cancer cells and releasing the synthesized drug protein.

[0050] In a preferred embodiment of the present invention, the natural ability of Salmonella to invade host animal cells is utilized to achieve bacterial invasion of host cancer cells.

[0051] Salmonella enterica is capable of invading and replicating within host cells, including epithelial cells and macrophages. This ability is embodied in the pathogenicity island 1 of Salmonella. Through the type III secretion system 1 (T3SS1), Salmonella secrete T3SS effector proteins, which induce extensive actin rearrangements within the host cell, leading to membrane ruffling and the formation of phagocytic vesicles containing the Salmonella. The type III secretion system 2 (T3SS2), encoded by the Salmonella pathogenicity island 2 (SPI-2) gene, then promotes the survival and proliferation of Salmonella within the phagocytic vesicles.

[0052] In a preferred embodiment of the present invention, the purpose of releasing the synthetic drug protein is achieved by using the listeriolysin-O (LLO) gene (SEQ ID No. 13) and protein sequence (SEQ ID No. 14) synthesized by Salmonella and optimized by Salmonella codons.

[0053] LLO is encoded by the hlyA gene of Listeria monocytogenes (LM) and can bind to the cholesterol in the host cell membrane to form a pore-like structure with a diameter of 35 nm.

[0054] LLO has a pH-sensitive acidic domain. The optimal pH for LLO precursor protein maturation and the onset of membrane perforation activity is approximately 5.0-5.5, while it is inactivated at neutral pH.

[0055] When Salmonella invades and forms phagocytic vesicles, they fuse with intracellular vacuoles such as lysosomes, significantly lowering the pH. Under these conditions, LLO activity is activated, exerting a perforating effect, allowing Salmonella to release the synthesized drug protein.

[0056] Therefore, in a preferred embodiment of the present invention, the membrane permeabilin gene is the listeriolysin hlyA gene.

[0057] In the present invention, the expression regulation of LLO has also been optimized. In US10987432B2, LLO is controlled by the promoter of the Salmonella pathogenicity island II sseA gene, inducing expression after the bacteria invade the cell and form phagocytic vesicles. However, its expression level and efficiency have a significant lag effect compared to the vector required for delivery. Therefore, in the present invention, the same T7 promoter is used to control LLO expression. This ensures that LLO expression is synchronized with the transcription and release of eukaryotic mRNA, maximizing overall release efficiency.

[0058] In order to carry out drug killing stably and efficiently, the present invention selects drug proteins with high efficiency and specific killing power, which can kill tumor cells while preventing accidental damage to other organs after drug protein leakage.

[0059] In a preferred embodiment of the present invention, the drug protein is the gene (SEQ ID No. 23) of the pyroptosis protein GSDMD-N (N segment of GSDMD) and the protein sequence of GSDMD-N (SEQ ID No. 24), and the gene (SEQ ID No. 25) of GSDME-N (N segment of GSDME) and the protein sequence of GSDME-N (SEQ ID No. 26).

[0060] In a preferred embodiment of the present invention, the drug protein can also be the gene (SEQ ID No.44) of the pyroptosis protein GSDMA-N (N segment fragment of GSDMA) and the protein sequence of GSDMA-N (SEQ ID No.45), the gene (SEQ ID No.46) of GSDMB-N (N segment fragment of GSDMB) and the protein sequence of GSDMB-N (SEQ ID No.47), and the gene (SEQ ID No.48) of GSDMC-N (N segment fragment of GSDMC) and the protein sequence of GSDMC-N (SEQ ID No.49).

[0061] In a preferred embodiment of the present invention, the expression vector further comprises a hypoxia-specific gene expression cassette comprising:

[0062] a) a positive hypoxia promoter, which is a promoter comprising an FNR binding site, and the positive hypoxia promoter can be induced to express under hypoxia;

[0063] b) genes essential for survival; and

[0064] c) an inverted hyperoxic promoter, which is a promoter comprising FNR and ArcA binding sites, and the inverted hyperoxic promoter can function under the oxygen content conditions of normal organs;

[0065] The essential gene for survival is a gene encoding alanine racemase.

[0066] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0067] In the present invention, "polynucleotide" refers to a macromolecule composed of multiple nucleotides linked by phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present invention can be codon-optimized for different host cells (e.g., Escherichia coli) to improve polypeptide expression. Methods for codon optimization are known in the art.

[0068] "Sequence identity" between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for assessing the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various known sequence analysis software. For example, sequence identity can be assessed using the EMBL-EBI online alignment tool (https: / / www.ebi.ac.uk / Tools / psa / ). The sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm using default parameters.

[0069] In order for bacteria to survive only in low oxygen concentrations, three requirements need to be met:

[0070] 1. Control the strength of the upstream promoter and background leakage expression.

[0071] If the oxygen sensor is partially leaky without binding to the upstream promoter, it will be impossible to regulate oxygen, resulting in the bacteria surviving at any oxygen concentration. Alternatively, if the upstream promoter is too weak, it will be unable to initiate the expression of downstream genes, resulting in the bacteria not surviving at any oxygen concentration.

[0072] 2. Select appropriate genes essential for survival.

[0073] By selecting genes essential for survival, it is possible to ensure that the bacteria die when the genes are not expressed, and to quickly ensure bacterial survival when the genes are induced by hypoxia;

[0074] 3. Under the switching conditions of low oxygen and high oxygen, the upstream promoter can quickly initiate transcription and quickly realize the synthetic expression of genes essential for survival, ensuring that the modified bacteria can survive. However, under high oxygen conditions, the expression of essential survival genes will not be initiated, resulting in bacterial death.

[0075] Therefore, in the present invention, a "hypoxia-specific gene expression cassette" refers to a segment of DNA that can initiate expression of an essential gene under hypoxic conditions, which contains an essential gene controlled by a hypoxia-inducible promoter and, if necessary, may further contain other regulatory elements required for the expression of the essential gene.

[0076] In the present invention, an "essential gene" refers to a gene that plays a crucial role in bacterial growth and / or survival. If the bacterium lacks this gene or its functional expression product, it will not survive, divide, and / or grow normally. Typical examples of bacteria lacking essential genes or their functional expression products are auxotrophic strains, which, in the absence of specific exogenous supplements, cannot survive, divide, and / or grow normally under in vitro culture conditions or in vivo environments. Essential genes are typically present in a single copy on the bacterial chromosome.

[0077] From this, we can see that the requirements for genes essential for survival are as follows:

[0078] 1. It is an essential gene for bacterial reproduction. If it is missing, it will lead to the rapid death of bacteria;

[0079] 2. The product of this gene does not exist in the normal environment or the human body, which ensures that it will not get out of control in the human environment. In addition, the corresponding expression product of this gene can be added to facilitate the cultivation and preparation of bacteria in a normal culture environment;

[0080] 3. This gene needs to be quickly activated under the regulation of the hypoxia promoter and can quickly synthesize products to realize the regulatory function of the host bacteria.

[0081] In the present invention, the essential gene for survival is a gene encoding alanine racemase.

[0082] The cell wall is an essential component of Gram-negative bacteria such as Escherichia coli and Salmonella. The core component of the cell wall is peptidoglycan. To synthesize peptidoglycan, bacteria require D-alanine, a crucial building block. Without D-alanine, bacteria cannot synthesize their cell wall and subsequently undergo cell lysis.

[0083] All amino acids exist in nature are L-type, so there are two genes in Gram-negative bacteria: the alr gene for the biosynthesis of alanine racemase and dadX, which are responsible for converting L-alanine into D-alanine to meet the needs of cell wall synthesis.

[0084] The study found that if the alr gene and dadX gene mutate at the same time, a lethal mutation of Salmonella can be achieved, and this mutation can be compensated by additional supplementation of D-alanine in the culture medium.

[0085] In our previous research, YB1 Salmonella used the asd gene as an essential gene for regulation. Compared to YB1 Salmonella, the present invention selected the alr gene and dadX gene as essential genes for survival. The alr gene and dadX gene are functionally homologous genes. Therefore, the alanine racemase gene in the present invention can be the alr gene (SEQ ID No. 27) and the protein sequence of Salmonella alr (SEQ ID No. 28) or the dadX gene (SEQ ID No. 29) and the protein sequence of Salmonella dadX (SEQ ID No. 30) from Salmonella; or the alr or dadX gene from other Gram-negative bacteria, or genes with equivalent functions.

[0086] In the present invention, the alanine racemase alr gene and dadX gene comprise nucleotide sequences having a sequence identity of greater than or equal to 81%, preferably greater than or equal to 82%, more preferably greater than or equal to 83%, greater than or equal to 84%, greater than or equal to 85%, greater than or equal to 86%, greater than or equal to 87%, greater than or equal to 88%, greater than or equal to 89%, greater than or equal to 90%, greater than or equal to 91%, greater than or equal to 92%, greater than or equal to 93%, greater than or equal to 94%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, or most preferably greater than or equal to 99% to the above-mentioned specific sequences.

[0087] In the present invention, alr gene knockout bacteria were first constructed, and then another gene, dadX, was modified, so that the bacteria became deficient in alr and dadX genes after editing. At the same time, a forward hypoxia promoter and a reverse hyperoxia promoter were used to regulate the additional alr or dadX gene.

[0088] The positive hypoxic promoter described herein is a hypoxic promoter regulated by FNR; the reverse hyperoxic promoter described herein is an antisense promoter negatively regulated by FNR and / or ArcA. The fumarate and nitrate reduction genes, fnr, are important genes regulating aerobic and anaerobic growth in Salmonella. This complex regulatory system has been extensively studied in Escherichia coli and Salmonella. The DNA-binding protein FNR encoded by the fnr gene senses changes in oxygen and controls the expression of various genes, achieving overall metabolic shifts. Therefore, DNA-binding sequences such as FNR and ArcA have become important mechanisms for controlling downstream gene expression.

[0089] FNR has an oxygen-sensitive [4Fe-4S] 2+ The FNR domain directly senses oxygen and regulates site-specific DNA binding. In contrast, ArcA senses signals from the aerobic respiratory chain. Therefore, Gram-negative facultative anaerobes such as Salmonella and Escherichia coli utilize two distinct mechanisms for sensing changes in oxygen concentration. FNR regulates gene expression in response to hypoxia, either through activation or repression.

[0090] To this end, the present invention uses the forward hypoxia promoters yhbU and ynfK that activate downstream gene expression under hypoxia by FNR, and the reverse hyperoxia promoters ydcI and cyoA that inhibit downstream gene expression under hypoxia by FNR and / or ArcA.

[0091] Among them, the positive hypoxia promoter used in the present invention is as follows:

[0092] 1. Salmonella yhbU promoter (yhbU-S) (SEQ ID No. 31), wherein the FNR binding site is "CTGCCTTAAATCAA";

[0093] 2. Escherichia coli ynfK promoter (ynfK-E) (SEQ ID No. 32), wherein the FNR binding site is "TTGCGCTATCTCAA";

[0094] Among them, the reverse hyperoxia promoter used in the present invention is as follows:

[0095] 1. Salmonella cyoA promoter (cyoA-S) (SEQ ID No. 33), which contains the FNR binding site "TTTATTGATAATAA" and the ArcA binding site "GTTAAGTA";

[0096] 2. Salmonella ydcI promoter (ydcI-S) (SEQ ID No. 34), which contains the FNR binding site "GTTATCAAAAACAA" and the ArcA binding site "GTTAATAA";

[0097] Through analysis of the above-mentioned reverse hypoxia promoter and forward hyperoxia promoter, we found that the FNR binding sites all conform to the "TTGATNNNNATCAA" pattern, and any base in the TTGAT and ATCAA sequences in the conserved binding sites can be replaced, but the total number does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; and ArcA conforms to the "GTTAATTA" pattern of its core region, and any base in the GTTAATTA sequence in the conserved binding site can be replaced, but the total number does not exceed 2.

[0098] Therefore, in a preferred embodiment of the present invention, the FNR binding site of the forward hypoxia promoter or the reverse hyperoxia promoter conforms to the pattern of TTGATNNNNATCAA, where N is any base of A, T, C, or G, and any base in the TTGAT or ATCAA sequence in the conserved binding site can be replaced, but the total number of replacements does not exceed 3, and three consecutive adjacent bases cannot be replaced; the ArcA binding site of the reverse hyperoxia promoter conforms to the pattern of GTTAATTA, wherein any base can be replaced, but the total number of replacements does not exceed 2.

[0099] In a more preferred embodiment of the present invention, the forward hypoxia promoter is selected from yhbU and ynfK; the essential survival gene is selected from alr and dadX; and the reverse hyperoxia promoter is selected from cyoA and ydcI.

[0100] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter cyoA.

[0101] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter ydcI.

[0102] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene dadX and the reverse hyperoxia promoter cyoA.

[0103] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene dadX and the reverse hyperoxia promoter ydcI.

[0104] The positive hypoxia promoter disclosed in patent CN104471057B is pepT. The pepT promoter is not completely regulated by FNR, but is a promoter that is dual-regulated by CRP-cAMP and FNR. Therefore, half of the FNR binding site in the pepT promoter is a CRP binding site, and half is a FNR binding site. The CRP-FNR binding region sequence of the pepT promoter is GTGACCTGACGCAA, of which the first half GTGA conforms to the first half of the CRP conserved binding site GTGANNNNNNTCAC, and the second half CGCAA conforms to the ATCAA portion of the FNR conserved region. The A at the tenth position is replaced by C, and the T at the eleventh position is replaced by G, which does not conform to the rules for positive hypoxia promoters in the present invention.

[0105] The FNR binding region of the reverse hyperoxia promoter sodA, disclosed in patent CN104471057B, contains only the first half of the FNR region, TTGAT, while the second half contains three consecutive substitutions of ATCAA with ATTTT, thus not containing a complete FNR binding site. The ArcA binding site is TTTAATTA, which, compared to the conserved core ArcA binding site 5'-GTTAATTA-3', has the first G replaced by a T. Therefore, the sodA promoter contains only a single ArcA binding site, and its FNR binding site is incomplete, which does not meet the requirements of the present invention for reverse hyperoxia promoters.

[0106] In summary, the present invention obtains a more efficient and safer hypoxia-specific gene expression cassette by screening a reasonable forward hypoxia promoter and a reverse hyperoxia promoter, and combining them with appropriate survival essential genes.

[0107] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is regulated by oxygen concentration.

[0108] For Gram-negative bacteria like Salmonella to survive in a hypoxic zone, an oxygen concentration of 1% is crucial, representing pathological hypoxia. This is because oxygen concentrations below 1% are a clear marker of a tumor hypoxic zone. In normal organs and tissues, there are no areas with oxygen concentrations below 1%. For example, in pancreatic cancer, cervical cancer, prostate cancer, and other tumors, the oxygen concentration in the hypoxic zone is less than 0.7%.

[0109] The hypoxia-specific gene expression cassette designed in this invention precisely regulates oxygen levels, enabling Salmonella and other Gram-negative bacteria to recognize and proliferate in hypoxic tumor areas. The goal of this invention is to enable the modified Salmonella and other Gram-negative bacteria to survive and proliferate in oxygen concentrations below 0.8%, and to achieve suicide lysis in normal oxygen concentrations.

[0110] Thus, in a more preferred embodiment of the present invention, the positive hypoxia promoter functions when the oxygen content is below 1%, but fails to function when the oxygen content is above 1%; and / or

[0111] The inverted hyperoxic promoter is functional when the oxygen content is above 1%, but is inactive when the oxygen content is below 1%.

[0112] In a further preferred embodiment of the present invention, the positive hypoxia promoter functions when the oxygen content is lower than 0.8%, but fails to function when the oxygen content is higher than 0.8%; and / or

[0113] The inverted hyperoxic promoter is functional when the oxygen content is above 0.8%, but is inactive when the oxygen content is below 0.8%.

[0114] In a preferred embodiment of the present invention, in the dual phage-derived promoter, one is sequentially linked to a ribosome binding site (RBS) (SEQ ID No. 35) and a membrane permeabilin gene, and the other is sequentially linked to an IRES sequence, a kozak sequence, an ORF region of a eukaryotic drug, a 3'-UTR region, and a sequentially expressed IRES sequence, a kozak sequence, an ORF region of a specific phage RNA polymerase gene, a polyA tail, and a T7 terminator sequence.

[0115] In a preferred embodiment of the present invention, the replication origin of the expression vector of the present invention is the high-copy replicon pUC (SEQ ID No. 36), or the low-copy replicon p15A, ColE1, or R6K.

[0116] Meanwhile, the expression vector of the present invention does not contain a resistance gene.

[0117] In a preferred embodiment of the present invention, the host bacteria of the expression vector are Gram-negative bacteria.

[0118] In the present invention, term " gram-negative bacteria " refers to after carrying out the known partial program of gram staining, does not retain the bacterium of the initial basic dye staining agent (such as, crystal violet) as.In exemplary gram staining, first by heating, cell is fixed on slide and dyed with basic dye (such as, crystal violet), and described basic dye is absorbed by both gram-negative bacteria and gram-positive bacteria.Then, slide is processed with mordant (such as, Gram's iodine solution), and described mordant is incorporated into basic dye (such as, crystal violet) and is trapped in cell.Then, by cell acetone or ethanol washing, then with the second dye of different colors counterstaining (such as, safranin).Gram-positive organism retains initial purple dyeing, and gram-negative organism decolorizes by organic washing solvent, therefore shows counterstaining. Exemplary Gram-negative bacteria include, but are not limited to, Escherichia species, Shigella species, Salmonella species, Campylobacter species, Neisseria species, Haemophilus species, Aeromonas species, Francisella species, Yersinia species, Klebsiella species, Bordetella species, Legionella species, Corynebacterium species, Citrobacter species, Chlamydia species, Brucella species, Pseudomonas species, Helicobacter species, and Vibrio species.

[0119] Thus, in a more preferred embodiment of the present invention, the Gram-negative bacteria are selected from the group consisting of Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simihuierlia, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, Paralytica, Vibrio sanguineus, Vibrio cholerae, Yersinia pestis, Coccus catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella catarrhalis, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolytica, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia and Helicobacter pylori.

[0120] In the present invention, the term "live bacteria" refers to a strain that has vitality, active nutritional metabolic characteristics, and is able to perform its own biological functions. Live bacteria can include bacterial biomass produced during the metabolic process of the strain.

[0121] In a more preferred embodiment of the present invention, the Gram-negative bacteria is Salmonella.

[0122] Another aspect of the present invention provides a method for a constitutive eukaryotic drug mRNA / DNA mixed dual delivery cytoplasmic expression system containing dual phage-derived promoters, comprising the following steps:

[0123] 1. Sequentially connect the phage-derived promoter, ribosome binding site and membrane-permeabilizing protein gene described in the present invention to construct a membrane-permeabilizing protein gene expression unit;

[0124] 2. Sequentially connect the phage-derived promoter IRES sequence, kozak sequence, eukaryotic drug ORF region, 3'-UTR region, and sequentially express the IRES sequence, kozak sequence, specific phage RNA polymerase gene ORF region, polyA tail, and T7 terminator sequence described in the present invention to construct a drug eukaryotic protein gene transcription unit and a unit that self-enhances in the eukaryotic cytoplasm.

[0125] Another aspect of the present invention provides a method for controlling drug mRNA expression in prokaryotic cells using the expression vector of the present invention, comprising the following steps:

[0126] 1. Prepare the expression vector of the present invention;

[0127] 2. Transform the constitutive expression promoter that controls the expression of the bacteriophage RNA polymerase gene into the chromosome of Gram-negative bacteria;

[0128] 3. Transform the expression vector of the present invention into Gram-negative bacteria.

[0129] When the expression vector of the present invention is introduced into Gram-negative bacteria (e.g., Salmonella typhimurium) as a therapeutic bacterium, the efficient expression of the drug protein gene and membrane-permeabilizing protein gene controlled by the specific phage RNA polymerase and the dual phage-derived promoter integrated into the bacterial chromosome will lead to increased bacterial toxicity. Therefore, in order to achieve safety as a therapeutic bacterium, the bacteria integrated with the expression vector of the present invention need to be modified by one or more attenuation methods. Attenuation methods include, but are not limited to, hypoxia-specific gene expression cassette regulation systems, nutritional deficiencies, stress response defects, and virulence island regulation defects.

[0130] Therefore, in a preferred embodiment of the present invention, the Gram-negative bacteria are attenuated Gram-negative bacteria.

[0131] In a more preferred embodiment of the present invention, the attenuated Gram-negative bacterium is Salmonella.

[0132] In a more preferred embodiment of the present invention, the attenuation method is selected from the group consisting of aroA gene deficiency of Salmonella and a hypoxia-specific gene expression cassette regulation system.

[0133] Another aspect of the present invention provides a modified Gram-negative bacterium comprising the expression vector of the present invention.

[0134] In a preferred embodiment of the present invention, the Gram-negative bacteria are selected from the group consisting of Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simihuierlia, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella Escherichia coli, Klebsiella pneumoniae, Vibrio parahaemolyticus, Vibrio cholerae, Yersinia pestis, Coccus catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia and Helicobacter pylori.

[0135] In a more preferred embodiment of the present invention, the Gram-negative bacteria is Salmonella.

[0136] Another aspect of the present invention provides use of the expression vector or the Gram-negative bacteria of the present invention in the preparation of tumor drugs.

[0137] In a preferred embodiment of the present invention, the tumor is a solid tumor.

[0138] The term "solid tumor" used in the present invention refers to an abnormal mass of tissue, generally not comprising a cyst or liquid area. Solid tumors may be benign (non-cancer), or malignant (cancer). Different types of malignant solid tumors are named after the cell type that forms them. Examples of malignant solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood cancer) does not form malignant solid tumors generally. Malignant solid tumors include but are not limited to the abnormal cell mass that may be derived from different tissue types, such as liver, colon, colorectal, skin, breast, pancreas, cervix uteri, uterine body, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testis, thyroid or lung etc., so malignant solid tumors include malignant solid liver tumors, colon tumors, colorectal tumors, skin tumors, breast tumors, pancreatic tumors, cervix tumors, uterine body tumors, bladder tumors, gallbladder tumors, kidney tumors, laryngeal tumors, lip tumors, oral cavity tumors, esophageal tumors, ovarian tumors, prostate tumors, stomach tumors, testicular tumors, thyroid tumors or lung tumors etc.

[0139] Thus, in a more preferred embodiment of the present invention, the solid tumor is selected from tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestine (colon cancer, rectal cancer), pelvic cavity (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer and bladder, etc.

[0140] In a further preferred embodiment of the present invention, the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

[0141] In the present invention, for the purpose of treatment, the term "subject" is preferably a subject in need of treatment for a target pathological condition, such as a tumor. For the purpose of prevention, the subject is preferably a subject at risk of developing a target pathological condition or prone to developing a target pathological condition. The term "subject" includes living organisms, such as prokaryotes and eukaryotes. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, hedgehogs, rats, and transgenic non-human animals. In a specific embodiment of the present invention, the subject is a human.

[0142] As used herein, "treatment" is a process for obtaining a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing the proliferation of neoplastic or cancerous cells (or destroying neoplastic or cancerous cells), inhibiting the metastasis of neoplastic cells, shrinking or reducing the size of a tumor, alleviating a malignant tumor, alleviating symptoms caused by a malignant tumor, improving the quality of life of a subject suffering from a malignant tumor, reducing the dosage of other drugs required to treat a malignant tumor, delaying the progression of a malignant tumor, curing a malignant tumor, and / or prolonging the survival of a patient suffering from a malignant tumor.

[0143] As used herein, an "effective amount" or "effective dose" of a bacterium, drug, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes that present during the course of disease. For therapeutic uses, beneficial or desired results include, for example, alleviating one or more symptoms of a disease (such as a tumor), reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, prolonging the survival of the treated subject, and / or delaying the progression of cancer in a patient. For example, an "effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in animal model systems that are predictive of efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Such an amount can be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0144] In some embodiments of the invention, the effective amount of bacteria in the bacteria, medicament or pharmaceutical composition comprises at least about 10 4 colony forming units (cfu), for example at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 cfu, including any ranges between listed values, such as 10 4 -10 8 cfu, 10 4 -10 9 cfu, 10 4 -10 10 cfu, 10 4 -10 11 cfu, 10 4 -10 12 cfu, 10 4 -10 13cfu, 10 5 -10 8 cfu, 10 5 -10 9 cfu, 10 5 -10 10 cfu, 10 5 -10 11 cfu, 10 5 -10 12 cfu, 10 5 -10 13 cfu, 10 6 -10 8 cfu, 10 6 -10​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​In some embodiments of the invention, the volume of an effective dose of the bacterium, medicament or pharmaceutical composition of the invention is less than or equal to about 3 ml, about 2.5 ml, about 2 ml, about 1.5 ml, about 1 ml, about 0.75 ml, about 0.5 ml, about 0.25 ml or about 0.1 ml. In some embodiments, the volume of an effective dose of the bacterium, medicament or pharmaceutical composition is about 20 ml, about 19 ml, about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml, about 13 ml, about 12 ml, about 11 ml, about 10 ml, about 9 ml, about 8 ml, about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml or about 1 ml. Optionally, the volume of the effective dose of the bacteria, drug or pharmaceutical composition is about 20.5 ml, about 19.5 ml, about 18.5 ml, about 17.5 ml, about 16.5 ml, about 15.5 ml, about 14.5 ml, about 13.5 ml, about 12.5 ml, about 11.5 ml, about 10.5 ml, about 9.5 ml, about 8.5 ml, about 7.5 ml, about 6.5 ml, about 5.5 ml, about 4.5 ml, about 3.5 ml, about 2.5 ml, about 1.5 ml, or about 0.5 ml. In some embodiments, the volume of an effective dose of the bacteria, drug or pharmaceutical composition is about 200 ml, about 190 ml, about 180 ml, about 170 ml, about 160 ml, about 150 ml, about 140 ml, about 130 ml, about 120 ml, about 110 ml, about 100 ml, about 90 ml, about 80 ml, about 70 ml, about 60 ml, about 50 ml, about 40 ml or about 30 ml, as well as ranges between any of the listed values, for example, 100 ml-110 ml, 100 ml-120 ml, 90 ml-120 ml, 90 ml-130 ml, etc. Optionally, the volume of the effective dose of the bacteria, drug or pharmaceutical composition is about 205 ml, about 195 ml, about 185 ml, about 175 ml, about 165 ml, about 155 ml, about 145 ml, about 135 ml, about 125 ml, about 115 ml, about 105 ml, about 95 ml, about 85 ml, about 75 ml, about 65 ml, about 55 ml, about 45 ml, about 35 ml or about 25 ml, and ranges between any of the listed values, such as 105 ml-115 ml, 105 ml-125 ml, 95 ml-125 ml, 95 ml-135 ml, etc.Optionally, the volume of an effective dose of the bacteria, drug or pharmaceutical composition is about 900 microliters, about 800 microliters, about 700 microliters, about 600 microliters, about 500 microliters, about 400 microliters, about 300 microliters, about 200 microliters, or about 100 microliters, optionally about 950 microliters, about 850 microliters, about 750 microliters, about 650 microliters, about 550 microliters, about 450 microliters, about 350 microliters, about 250 microliters, about 150 microliters, or about 50 microliters.

[0146] In some embodiments, the effective dose of the bacterium, drug, or pharmaceutical composition has a volume of less than or equal to about 2.0 ml.

[0147] The dosage ranges mentioned herein are merely exemplary and do not limit the dosage ranges that can be selected by a physician. The amount of the active ingredient (e.g., the bacteria of the present invention) in the pharmaceutical compositions of the present invention can vary depending on factors such as the individual's disease state, age, sex, and weight, including the presence or absence of a tumor, the type of tumor being treated, the severity of the tumor, the activity or viability of the bacteria, drug, or pharmaceutical composition, the route of administration, the duration of treatment, the drugs used in combination with the bacteria, drug, or pharmaceutical composition (if any), the subject's diet and general health, and similar factors well known in the art. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased depending on the urgency of the therapeutic situation.

[0148] As used herein, a "pharmaceutically acceptable carrier" includes any material that allows the ingredient to retain biological activity when combined with the active ingredient and does not react with the subject's immune system, including but not limited to disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants, or lubricants. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion). For example, depending on the route of administration, the bacteria of the present invention can be wrapped in a material to protect the bacteria from the effects of acids and other natural conditions that can inactivate the bacteria. Pharmaceutically acceptable carriers include physiological saline, PBS buffer, sterile aqueous solutions or dispersions, and powders for the temporary preparation of injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any incompatibility with the active compound, may be in the pharmaceutical composition of the present invention.

[0149] Therefore, another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the expression vector of the present invention or the modified bacteria of the present invention. In one embodiment, the modified bacteria are live bacteria.

[0150] In a preferred embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0151] In a more preferred embodiment of the present invention, the pharmaceutically acceptable carrier is selected from disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants and lubricants.

[0152] In a preferred embodiment of the present invention, the pharmaceutical composition is used to treat solid tumors. The bacteria, drugs, or pharmaceutical compositions of the present invention are administered by intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0153] In a preferred embodiment of the present invention, the bacteria, drugs or pharmaceutical compositions of the present invention can be formulated for administration by the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0154] The dosage form of the drug or pharmaceutical composition of the present invention can be a solution, emulsion, freeze-dried preparation or suspension; for oral administration, the dosage form can be a tablet or capsule; for intranasal dosage form, the dosage form can be a powder, nasal drops or aerosol form; for topical administration, the dosage form can be an aqueous solution, suspension, ointment, cream or gel; for rectal or vaginal administration, the dosage form can be a suppository, enema or delivered as part of an endoscopic or colonoscopy procedure.

[0155] Bacteria, medicine or pharmaceutical compositions of the present invention can be manufactured by methods well known in the art, such as growing the microorganisms in a fermentation tank, followed by centrifugal concentration and washing, filtering or dialysis, conventional granulation, mixing, dissolving, encapsulating, lyophilizing or emulsifying processes and other methods. Bacteria, medicine or pharmaceutical compositions of the present invention can be produced in various forms, including particles, precipitations or microparticles, powders, including freeze-dried, rotary-dried or spray-dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. Preparations can optionally contain stabilizers, pH adjusting agents, surfactants, bioavailability regulators and combinations thereof.

[0156] Bacteria, medicine or pharmaceutical composition of the present invention can be used alone or in combination with other compounds or compositions in the presence of a carrier. In a preferred embodiment of the present invention, the bacterium, medicine or pharmaceutical composition can be used in combination with other malignant tumor therapies (including but not limited to, radiotherapy, chemotherapy and surgical operation). The bacterium, medicine or pharmaceutical composition can be used as an adjuvant in therapy in such a case.

[0157] In another aspect, the present invention provides a method for treating tumors, comprising administering the eukaryotic expression vector of the present invention, the Gram-negative bacteria of the present invention, or the pharmaceutical composition of the present invention to a subject.

[0158] In one embodiment, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition is used to treat tumors. In one embodiment, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition is used to induce an anti-tumor specific immune response in a subject with a tumor. In one embodiment, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition is used to induce anti-tumor immune memory in a subject with a tumor. In one embodiment, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition is used to prevent or treat metastasis or recurrence of a tumor. In one embodiment, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition is used to treat a tumor that has become resistant to or failed previous anti-tumor therapy.

[0159] In one embodiment of the present invention, the tumor is a tumor of the nervous system, respiratory system, digestive system, urinary system, reproductive system, hematopoietic system, lymphatic system, endocrine system, or skin and mucosa. In one embodiment, the tumor is a sarcoma or carcinoma. In one embodiment, the tumor is a solid tumor.

[0160] In a further preferred embodiment of the present invention, the solid tumor is selected from the group consisting of breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal (colon cancer, rectal cancer), pelvic (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer and bladder tumors / cancers, etc.

[0161] In a more preferred embodiment of the present invention, the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

[0162] The method for treating tumors provided by the present invention comprises administering an effective amount of the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor.

[0163] In an embodiment of the present invention, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention can be administered by the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0164] In some embodiments of the present invention, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention can be administered at intervals of, for example, about 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1 day, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including ranges between any two of the listed values, for example, 1 minute to 10 minutes, 1 minute to 30 minutes, 1 minute to 1 hour, 1 minute to 2 hours, 1 minute to 4 hours, 1 minute to 12 hours, 1 minute to 18 hours, or 10 days. -1 day, 10 minutes - 30 minutes, 10 minutes - 1 hour, 10 minutes - 2 hours, 10 minutes - 4 hours, 10 minutes - 12 hours, 10 minutes - 18 hours, 10 minutes - 1 day, 30 minutes - 1 hour, 30 minutes - 2 hours, 30 minutes - 4 hours, 30 minutes - 12 hours, 30 minutes - 18 hours, 30 minutes - 1 day, 30 minutes - 2 days, 1 hour - 2 hours, 1 hour - 4 hours, 1 hour - 12 hours, 1 hour - 18 hours, 1 hour - 1 day, 4 hours - 12 hours, 4 hours - 18 hours, 4 hours - 1 day, 1 day - 2 days, 1 day - 3 days, 1 day - 4 days, 1 day - 5 days, 1 day - 7 days, 1 day - 10 days, 2 days - 3 days, 2 days - 4 days, 2 days - 5 days, 2 days - 7 days, 2 days - 10 days, or 5 days - 10 days. In some embodiments, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks, or once every twenty-six weeks.In some embodiments, the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition is formulated, for example, to be administered at intervals of about 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1 day, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including ranges between any two of the listed values, for example, 1 minute to 10 minutes, 1 minute to 30 minutes, 1 minute to 1 hour, 1 minute to 2 hours, 1 minute to 4 hours, 1 minute to 12 hours, 1 minute to 18 hours, or 1 minute to 24 hours. -1 day, 10 minutes - 30 minutes, 10 minutes - 1 hour, 10 minutes - 2 hours, 10 minutes - 4 hours, 10 minutes - 12 hours, 10 minutes - 18 hours, 10 minutes - 1 day, 30 minutes - 1 hour, 30 minutes - 2 hours, 30 minutes - 4 hours, 30 minutes - 12 hours, 30 minutes - 18 hours, 30 minutes - 1 day, 30 minutes - 2 days, 1 hour - 2 hours, 1 hour - 4 hours, 1 hour - 12 hours, 1 hour - 18 hours, 1 hour - 1 day, 4 hours - 12 hours, 4 hours - 18 hours, 4 hours - 1 day, 1 day - 2 days, 1 day - 3 days, 1 day - 4 days, 1 day - 5 days, 1 day - 7 days, 1 day - 10 days, 2 days - 3 days, 2 days - 4 days, 2 days - 5 days, 2 days - 7 days, 2 days - 10 days, or 5 days - 10 days. In some embodiments, the vector, bacteria, drug or pharmaceutical composition is formulated to be administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks or once every twenty-six weeks. The dosage regimen may depend on the pattern of pharmacokinetic decay that the practitioner expects to achieve. The progress of the therapy can be monitored by conventional techniques and assays. The dosage regimen can vary over time.

[0165] The present invention also provides a method for inducing an anti-tumor specific immune response in a subject having a tumor, comprising administering an effective amount of the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to the subject having a tumor.

[0166] The present invention also provides a method for inducing anti-tumor immune memory in a subject having a tumor, comprising administering an effective amount of the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to the subject having a tumor.

[0167] The present invention also provides a method for preventing or treating tumor metastasis or recurrence, comprising administering an effective amount of the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor.

[0168] The present invention also provides a method for preventing or treating tumor metastasis or recurrence, comprising administering an effective amount of the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from tumor metastasis or recurrence or a subject at high risk of tumor metastasis or recurrence.

[0169] The present invention also provides a method for treating a tumor that has become resistant to or failed to be treated with previous anti-tumor therapy, comprising administering an effective amount of the eukaryotic expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor that has become resistant to or failed to be treated with previous anti-tumor therapy.

[0170] The present invention achieves the following beneficial effects by adopting the above technical solution:

[0171] 1. The present invention combines a phage-derived promoter with a specific phage RNA polymerase gene to coordinate the two, and uses a plasmid-based expression platform to achieve stable and efficient expression of drug proteins in the tumor area.

[0172] 2. The present invention combines a phage-derived promoter with a membrane-permeabilizing protein gene and a eukaryotic drug gene mRNA / DNA mixed vector, so that the three cooperate to achieve stable and efficient drug delivery, realize a true cytoplasmic expression system, and thus achieve efficient killing of tumor cells.

[0173] 3. The present invention rationally designs a hypoxia-specific gene expression cassette, which reduces the overall mutation rate of the modified bacteria, achieves specific distribution within the tumor, and accelerates clearance in normal organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0174] Figure 1. Agarose gel electrophoresis identification and verification of the positive hypoxia promoter yhbU-S and ynfK-S clones.

[0175] Figure 2. Amplification verification diagram of essential gene library identified by agarose gel electrophoresis.

[0176] Figure 3 Agarose gel electrophoresis identification of reverse hyperoxia promoter cyoA-S and ydcI-S clone verification.

[0177] Figure 4 Schematic diagram of the hypoxia-specific gene expression cassette.

[0178] Fig. 5 Schematic diagram of the lambda RED recombinase and CRE recombinase systems.

[0179] Figure 6 shows the knockout experiment and identification of the Salmonella aroA gene.

[0180] (A) Agarose gel electrophoresis to identify the PCR amplification product of the aroA gene knockout fragment;

[0181] (B) Verification of the PCR amplification product of the target fragment of strain SWT003 identified by agarose gel electrophoresis.

[0182] Figure 7. Knockout experiment and identification of Salmonella alr.

[0183] (A) Agarose gel electrophoresis to identify the PCR amplification product of the alr gene knockout fragment;

[0184] (B) Verification of the PCR amplification product of the target fragment of strain SWT004 identified by agarose gel electrophoresis.

[0185] FIG8 is a verification diagram of strains SWT1001 and SWT1005 after insertion of the hypoxia-specific gene expression cassette (alr is an essential gene), as identified by agarose gel electrophoresis.

[0186] FIG9 is a diagram showing the verification of the PCR amplification product of SWT007 identified by agarose gel electrophoresis.

[0187] FIG10 is a verification diagram of strains SWT2009 and SWT2013 after insertion of the hypoxia-specific gene expression cassette (dadX is an essential gene) as identified by agarose gel electrophoresis.

[0188] FIG11 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1001.

[0189] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0190] (B) Aerobic culture on LB plates without D-alanine.

[0191] FIG12 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1005.

[0192] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0193] (B) Aerobic culture on LB plates without D-alanine.

[0194] Figure 13. Verification of oxygen adaptability of strains SWT2009 and SWT2013.

[0195] (A) SWT2009 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0196] (B) SWT2013 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0197] FIG14 shows the results of the oxygen adaptability verification test of various Salmonella strains at an oxygen concentration below 0.8%.

[0198] (A) SWT1001 was cultured on LB plates;

[0199] (B) SWT1005 was cultured on LB plates;

[0200] (C) SWT2009 was cultured on LB plates;

[0201] (D) SWT2013 was cultured on LB plates.

[0202] Figure 15 is a schematic diagram of the pPRO005 cassette containing the chloramphenicol resistance gene, lacUV5 and T7 RNA polymerase genes.

[0203] Figure 16: Enzyme digestion identification of plasmid pPRO005 (the arrow indicates the excised T7 RNA polymerase gene cassette fragment).

[0204] Figure 17 Identification diagram of the recombination of the T7 RNA polymerase constitutive expression cassette controlled by lacUV5 into the Salmonella chromosome.

[0205] (A) PCR amplified fragment used for recombination (primers SWTO102 and SWTO103);

[0206] (B) PCR identification diagrams of the constructed SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P strains.

[0207] Figure 18 is a schematic diagram of the construction and function of the plasmid vector pEU011.

[0208] (A) Construction diagram of plasmid vector pEU011;

[0209] (B) Schematic diagram of the function of plasmid vector pEU011.

[0210] Figure 19 shows PCR identification of plasmid vector pEU011.

[0211] Figure 20 is a schematic diagram of the construction and function of the plasmid vector pEU010.

[0212] (A) Construction diagram of plasmid vector pEU010;

[0213] (B) Schematic diagram of the functioning process of plasmid vector pEU010.

[0214] Figure 21 shows the PCR identification of plasmid vector pEU010.

[0215] Figure 22 is a schematic diagram of the construction and function of the plasmid vector pIKDE-EGFP.

[0216] (A) Construction diagram of plasmid vector pIKDE-EGFP;

[0217] (B) Schematic diagram of the function of the plasmid vector pIKDE-EGFP.

[0218] FIG23 is fluorescence microscopy observation of the green fluorescence expression intensity after BSR-T7 / 5 stably expressing T7 polymerase was transfected with pEU010, pEU011, and pIKDE-EGFP plasmids.

[0219] (A) BSR-T7 / 5 cell control, white light image on top, fluorescence image on bottom;

[0220] (B) BSR-T7 / 5 cells transfected with pEU010, the upper part is the white light image, the lower part is the fluorescence image;

[0221] (C) BSR-T7 / 5 cells transfected with pEU011, the upper part is the white light image, the lower part is the fluorescence image;

[0222] (D) BSR-T7 / 5 cells were transfected with pIKDE-EGFP. The upper part shows the white light image, and the lower part shows the fluorescence image.

[0223] Figure 24 Flow cytometric analysis of the proportion of green fluorescent cells after BSR-T7 / 5 cells stably expressing T7 polymerase were transfected with pEU010, pEU011, and pIKDE-EGFP plasmids.

[0224] (A) pEU010 transfected BSR-T7 / 5 cells;

[0225] (B) pEU011 transfected BSR-T7 / 5 cells;

[0226] (C) BSR-T7 / 5 cells were transfected with pIKDE-EGFP.

[0227] Figure 25 is a schematic diagram of the construction of plasmid pSWT004 used to construct YB1-like Salmonella SWT005.

[0228] FIG26 is a diagram showing the verification of enzyme digestion products of plasmid pSWT004 identified by agarose gel electrophoresis.

[0229] Figure 27 is a diagram showing verification of the PCR amplification product of the cm-pepT-asd-sodA fragment identified by agarose gel electrophoresis.

[0230] FIG28 is a diagram showing verification of PCR amplification products of Salmonella SWT005 identified by agarose gel electrophoresis.

[0231] Figure 29 shows the results of the oxygen adaptability verification test of YB1-like Salmonella SWT005.

[0232] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0233] (B) Cultured under aerobic conditions on LB plates without D-alanine.

[0234] Figure 30 shows PCR identification of strain SWT008.

[0235] Figure 31 Schematic diagram of plasmid vector pPRO013.

[0236] Figure 32 shows PCR identification of strain SWT5025.

[0237] FIG33 compares the growth ability of strains SWT5015, SWT5015C, and SWT5025 in culture medium.

[0238] (A) Growth curves of strains SWT5015, SWT5015C, and SWT5025 in liquid LB medium;

[0239] (B) Count analysis of strains SWT5015, SWT5015C, and SWT5025 at the same OD on solid LB plates.

[0240] Figure 34 Identification of EGFP mRNA transcription in SWT5015, SWT5015C and SWT5025 strains.

[0241] (A) Agarose gel electrophoresis to identify total RNA extracted from the control strain SWT1005 and strains SWT5015, SWT5015C, and SWT5025;

[0242] (B) Reverse transcription qPCR and non-reverse transcription qPCR were used to amplify EGFP RNA of the control strain SWT1005 and strains SWT5015, SWT5015C, and SWT5025, respectively.

[0243] Figure 35 Western Blot identification of the expression of T7 RNA polymerase and LLO in strains SWT5025, SWT5015C and SWT5025.

[0244] (A) T7 RNA polymerase expression in strains SWT5025, SWT5015C, and SWT5025 under LB medium and N-salts medium conditions;

[0245] (B) LLO expression in strains SWT5025, SWT5015C, and SWT5025 under LB medium and N-salts medium conditions.

[0246] FIG36 compares the EGFP expression efficiency of the strains SWT5015C, SWT5015, and SWT5025 after invading cancer cells.

[0247] (A) EGFP expression in cells after invasion was observed under a fluorescence microscope;

[0248] (B) The fluorescence signal of EGFP was analyzed on a microplate reader.

[0249] Figure 37 shows PCR identification of plasmid vectors pEU013 and pEU014.

[0250] Figure 38 qPCR identification of target gene mRNA transcription in SWT5115, SWT5119, SWT5215, and SWT5219. The Cq value represents the amplification threshold.

[0251] (A) GSDMD-N mRNA was amplified in the control strain SWT1005 and SWT5115 using reverse transcription qPCR and non-reverse transcription qPCR, respectively;

[0252] (B) GSDME-N mRNA was amplified in the control strain SWT1005 and SWT5215 using reverse transcription qPCR and non-reverse transcription qPCR, respectively;

[0253] (C) GSDMD-N mRNA was amplified in the control strains SWT1005 and SWT5119 using reverse transcription qPCR and non-reverse transcription qPCR, respectively;

[0254] (D) GSDME-N mRNA was amplified in the control strains SWT1005 and SWT5219 using reverse transcription qPCR and non-reverse transcription qPCR, respectively.

[0255] Figure 39 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT1005 as the control, SWT5115, and SWT5215 after co-culture with cancer cells under anaerobic conditions.

[0256] (A) EMT6;

[0257] (B)MFC;

[0258] (C)RM-1.

[0259] Figure 40 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT1005 as the control, SWT5115, and SWT5215 after co-culture with cancer cells under anaerobic conditions.

[0260] (D) SCC7;

[0261] (E)CT26;

[0262] (F)MB49.

[0263] Figure 41 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT1005 as the control, SWT5115, and SWT5215 after co-culture with cancer cells under anaerobic conditions.

[0264] (G)A549;

[0265] (H) Neuro-2a;

[0266] (I)ID8.

[0267] Figure 42 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT1005 as the control, SWT5115, and SWT5215 after co-culture with cancer cells under anaerobic conditions.

[0268] (J) Hepa1-6;

[0269] (K) Renca;

[0270] (L)Pan02.

[0271] Figure 43 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT1005 as the control, SWT5115, and SWT5215 after co-culture with cancer cells under anaerobic conditions.

[0272] (M)SK-MEL-5;

[0273] (N)K7M2.

[0274] Figure 44 shows the killing effect of CCK8 on EMT6, K7M2, Hepa1-6, A549, SK-MEL-5, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7 and MB49 cancer cells.

[0275] Figure 45 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT2009 as the control, SWT5119, and SWT5219 after co-culture with cancer cells under anaerobic conditions.

[0276] (A) EMT6;

[0277] (B)MFC;

[0278] (C)RM-1.

[0279] Figure 46 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT2009 as the control, SWT5119, and SWT5219 after co-culture with cancer cells under anaerobic conditions.

[0280] (D) SCC7;

[0281] (E)CT26;

[0282] (F)MB49.

[0283] Figure 47 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT2009 as the control, SWT5119, and SWT5219 after co-culture with cancer cells under anaerobic conditions.

[0284] (G)A549;

[0285] (H) Neuro-2a;

[0286] (I)ID8.

[0287] Figure 48 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT2009 as the control, SWT5119, and SWT5219 after co-culture with cancer cells under anaerobic conditions.

[0288] (J) Hepa1-6;

[0289] (K) Renca;

[0290] (L)Pan02.

[0291] Figure 49 shows the microscopic images of the killing of cells by pyroptosis caused by the strains SWT2009 as the control, SWT5119, and SWT5219 after co-culture with cancer cells under anaerobic conditions.

[0292] (M)SK-MEL-5;

[0293] (N)K7M2.

[0294] Figure 50 shows the killing effects of CCK8 on EMT6, K7M2, A549, SK-MEL-5, MFC, and SCC7 cancer cells using the control strain SWT2009 and the expression vector strains SWT5119 and SWT5219.

[0295] Figure 51 shows the identification of the inhibitory effects of expression vector strains SWT5115, SWT5215 and control strain SWT1005 in different mouse tumor models (vehicle is a blank control without drug treatment).

[0296] (A) EMT6;

[0297] (B)K7M2;

[0298] (C) Hepa1-6;

[0299] (D)A549.

[0300] Figure 52 shows the identification of the inhibitory effects of expression vector strains SWT5115, SWT5215 and control strain SWT1005 in different mouse tumor models (vehicle is a blank control without drug treatment).

[0301] (A)B16F10;

[0302] (B) Renca;

[0303] (C)MFC;

[0304] (D)Pan02.

[0305] Figure 53 shows the identification of the inhibitory effects of expression vector strains SWT5115, SWT5215 and control strain SWT1005 in different mouse tumor models (vehicle is a blank control without drug treatment).

[0306] (A)RM-1;

[0307] (B) CT26;

[0308] (C) ID8;

[0309] (D) Neuro-2a.

[0310] Figure 54 shows the identification of the inhibitory effects of expression vector strains SWT5115, SWT5215 and control strain SWT1005 in different mouse tumor models (vehicle is a blank control without drug treatment).

[0311] (A) SCC7;

[0312] (B)MB49. DETAILED DESCRIPTION

[0313] The present application will be further described in detail below through the accompanying drawings and examples, through which the features and advantages of the present application will become more clear and distinct.

[0314] Example 1: Construction of a hypoxia-specific gene expression cassette with alr and dadX genes as essential genes for survival

[0315] In this example, the forward hypoxia promoter used Salmonella yhbU (yhbU-S) (SEQ ID No. 31), the survival essential gene used Salmonella alr gene (SEQ ID No. 27), the reverse hyperoxia promoters used Salmonella cyoA (cyoA-S) (SEQ ID No. 33) and Salmonella ydcI (ydcI-S) (SEQ ID No. 34), and the forward hypoxia promoter used Escherichia coli ynfK (ynfK-E) (SEQ ID No. 32), the survival essential gene used Salmonella dadX gene (SEQ ID No. 29), and the reverse hyperoxia promoters used Salmonella cyoA (cyoA-S) (SEQ ID No. 33) and Salmonella ydcI (ydcI-S) (SEQ ID No. 34) to construct a hypoxia-specific gene expression cassette.

[0316] The combination of hypoxia-specific gene expression cassettes is shown in Table 1.

[0317] Table 1 Combinations of forward hypoxia promoter and reverse hyperoxia promoter

[0318] (The essential genes for survival are the alr gene or dadX gene of Salmonella)

[0319] (I) Construction of a positive hypoxia promoter clone library

[0320] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Industrial Microbiological Culture Collection) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) and inoculate it into 5 ml LB liquid medium and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The OD 600 The reading is between 2-3;

[0321] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium and mix it into the corresponding primer and high-fidelity PCR amplification enzyme (purchased from TAKARA) system, and then put the mixture into the PCR amplification device.

[0322] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes;

[0323] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0324] 4. Mix the recovered product with the endonuclease (purchased from NEB) digestion system and incubate at 37°C for 1 hour;

[0325] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0326] The primers and endonucleases used for different forward hypoxia promoters are as follows:

[0327] 1. yhbU-S (SEQ ID No. 31): strain SWT001 was amplified using primers SWTO19 and 20, and the product was double-cleaved with NotI and HindIII and recovered;

[0328] 2. ynfK-E (SEQ ID No. 32): amplified from E. coli strain DH10B using primers SWTO21, 22, and the product was double-cleaved with NotI and HindIII and recovered;

[0329] The results of verification of the forward hypoxia promoter clone library by agarose gel electrophoresis are shown in FIG1 .

[0330] (2) Construction of a gene library essential for survival

[0331] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC, China Industrial Microbiology Culture Collection) and inoculate it into 5 ml of LB liquid medium. Incubate the culture in a shaker at 37°C and 220 rpm for 16 hours.

[0332] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium and mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then place the mixture into the PCR amplification device.

[0333] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes;

[0334] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0335] 4. Mix the recovered product with the enzyme digestion system and incubate at 37 degrees for 1 hour;

[0336] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0337] The essential genes for survival from different sources are as follows:

[0338] 1. Salmonella alr gene (SEQ ID No. 27): strain SWT001 was amplified using primers SWTO35 and 36, and the product was double-cut with HindIII and XhoI and recovered;

[0339] 2. Salmonella dadX gene (SEQ ID No. 29): strain SWT001 was amplified using primers SWTO39 and 40, and the product was double-cut with HindIII and XhoI and recovered;

[0340] The results of verification of the essential survival gene library by agarose gel electrophoresis are shown in FIG2 .

[0341] (III) Construction of the reverse hyperoxia promoter library

[0342] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC, China Industrial Microbiology Culture Collection) and inoculate it into 5 ml of LB liquid medium. Incubate it in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0343] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium and mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then place the mixture into the PCR amplification device.

[0344] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes;

[0345] 3. The amplified PCR product is purified and recovered through a DNA gel recovery system;

[0346] 4. Mix the recovered product with the enzyme digestion system and incubate at 37°C for 1 hour;

[0347] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0348] The primers and endonucleases used for different reverse hyperoxia promoters are as follows:

[0349] 1. cyoA-S (SEQ ID No. 33): strain SWT001 was amplified using primers SWTO43 and 44, and the product was double-cleaved with XhoI and PstI and recovered;

[0350] 2. ydcI-S (SEQ ID No. 34): strain SWT001 was amplified using primers SWTO51 and 52, and the product was double-cut with XhoI and PstI and recovered;

[0351] The results of verifying the reverse high oxygen promoter clone library by agarose gel electrophoresis are shown in FIG3 .

[0352] (IV) Construction of a combinatorial library of forward hypoxia promoters, essential survival genes, and reverse hyperoxia promoters

[0353] The composition of the hypoxia-specific gene expression cassette is shown in FIG4 , which is composed of a forward hypoxia promoter, a survival-essential gene, and a reverse hyperoxia promoter in sequence.

[0354] As shown in Table 1, a cloned library was formed by combining sequences of the forward hypoxia promoter library digested with NotI and HindIII, the survival essential gene Salmonella alr gene (SEQ ID No. 27) or Salmonella dadX gene (SEQ ID No. 29) digested with HindIII and XhoI, and the reverse hyperoxia promoter library digested with XhoI and PstI, and ligated into the plasmid pSWT003 vector (vector backbone, pBlueScript SK (+), purchased from Biowind) digested with SpeI and PstI, and the plasmid pSWT007 vector (containing double-sided same-direction loxP sequences and a DNA fragment of the chloramphenicol resistance gene (SEQ ID No. 37)) digested with SpeI and NotI.

[0355] The products amplified from Salmonella SWT001 or Escherichia coli DH10B using the above primers and recovered by enzyme digestion were combined and ligated by enzyme ligation reaction, transformed into DH10B bacteria and coated on LB plates containing 25 μg / ml chloramphenicol to obtain the corresponding plasmids shown in Table 1.

[0356] (V) Construction of the strain corresponding to the present invention

[0357] 1. Wild-type Salmonella Typhimurium (SWT001)

[0358] Wild-type Salmonella typhimurium (SWT001) was purchased from China Industrial Microbiology Culture Collection Center (CICC).

[0359] 2. Construction of Salmonella (SWT002) containing temperature-inducible lambda-RED recombinase and loxp-CRE enzyme systems

[0360] The Lambda-RED recombination system is widely used in homologous recombination of Gram-negative bacteria. In the present invention, this system is composed of plasmid pSWT001.

[0361] Plasmid pSWT001, shown in Figure 5, contains a lambda-RED recombinase module (SEQ ID No. 38) (similar in function to the plasmid vector psim6 from Biowind) and a loxP-Cre recombinase module (SEQ ID No. 38) (similar in function to the plasmid vector 705-Cre from Gene Bridges). The lambda-RED recombinase module consists of three recombinases: EXO, BET, and GAM. These recombinases are controlled by the CI857 temperature regulator. Therefore, expression is lost at 32°C and is only activated at temperatures above 37°C. Under temperature-induced conditions, the recombinase EXO cleaves the 5' end of double-stranded linear DNA, creating a single-stranded DNA overhang at the 3' end. This single-stranded DNA is bound by the BET protein and protected from degradation by other nucleases. GAM inhibits endogenous bacterial nucleases. Homologous arms for homologous recombination are approximately 35-50 bp and are added by PCR to flank the DNA fragment to be recombined. The advantage of this technology is that it can accurately target the target area of ​​the bacterial chromosome without causing additional mutations. The outermost parts of the recombinant double-stranded DNA fragments include homologous arm sequences of the position where recombination is required, which contain loxp sequences in the same direction on both sides, totaling 34bp. The sites of the double loxp in the same direction are chloramphenicol resistance genes, which are used to screen recombinant bacteria. After successful recombination, the CRE enzyme system carried on the pSWT001 plasmid (also controlled by the CI857 temperature regulator) specifically recognizes the loxp sequence and can cut the sequence in the middle of the loxp in the same direction, leaving a loxp sequence, thereby eliminating the chloramphenicol resistance gene. Repeating the above operations can achieve continuous knockout and knock-in of genes.

[0362] The specific steps are as follows:

[0363] (1) Streak the strain SWT001 on an LB plate and culture it in a 37°C incubator overnight;

[0364] (2) Pick a single clone and inoculate it into 5 ml of LB liquid medium and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0365] (3) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3 Place on ice and let stand for 1 hour;

[0366] (4) Wash the cells three times with sterilized purified water;

[0367] (5) The recovered cells were mixed with 10 ng of plasmid pSWT001 and electroporated at a voltage of 1.8 kV;

[0368] (6) The electroporated cells were spread on LB plates containing 100 μg / ml ampicillin sodium and cultured overnight in a 32°C constant temperature incubator until a single clone colony was grown, named SWT002.

[0369] 3. Construction of attenuated Salmonella with aroA gene knockout (SWT003)

[0370] (1) A single clone of SWT002 was picked and inoculated into 5 ml of LB liquid culture medium containing 100 μg / ml ampicillin sodium, and placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0371] (2) Inoculate the culture into fresh LB liquid medium containing 100 μg / ml ampicillin sodium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0372] (3) Wash the cells three times with sterilized purified water.

[0373] (4) Prepare PCR products of SWTO1 and SWTO2.

[0374] Primers SWTO1 and SWTO2 were mixed with plasmid pSWT002 (containing loxP sequences in the same direction on both sides and a chloramphenicol resistance gene SEQ ID No. 39 in the middle), and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0375] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0376] The amplified PCR products were subjected to agarose gel electrophoresis to verify the PCR amplification products of SWTO1 and SWTO2 of the aroA gene knockout fragments. The verification results are shown in Figure 6A.

[0377] The PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were measured using nanodrop.

[0378] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO1 and SWTO2 and electroporated at a voltage of 1.8 kV.

[0379] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0380] (7) Positive clones were identified by colony PCR, and the insertion of chloramphenicol resistance gene was identified using SWTO4, SWTO6, SWTO3 and SWTO5.

[0381] The target fragment PCR amplification product of strain SWT003 was verified by agarose gel electrophoresis, and the verification results are shown in Figure 6B.

[0382] (8) The positive single clone was inoculated into 5 ml of LB medium and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0383] 4. Construction of alr gene knockout attenuated bacteria (SWT004)

[0384] (1) A single clone of SWT003 was selected and inoculated into 5 ml of LB liquid culture medium, and then placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0385] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0386] (3) Wash the cells three times with sterilized purified water.

[0387] (4) Preparation of PCR products of SWTO70 and SWTO71.

[0388] Primers SWTO70 and SWTO71 were mixed with plasmid pSWT002 and a high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0389] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0390] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0391] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO70 and SWTO71 and electroporated at a voltage of 1.8 kV.

[0392] The PCR amplification products of SWTO70 and SWTO71 of the alr gene knockout fragments were verified by agarose gel electrophoresis. The verification results are shown in Figure 7A.

[0393] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0394] (7) Positive clones were identified by colony PCR, and SWTO72, SWTO5, SWTO6, and SWTO73 were used to identify the insertion of the chloramphenicol resistance gene.

[0395] The PCR amplification product of the target fragment of strain SWT004 was verified by agarose gel electrophoresis, and the verification results are shown in Figure 7B (the primers used were SWTO72, SWTO5, SWTO6, and SWTO73).

[0396] (8) The positive single clone was inoculated into 5 ml of LB medium and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0397] Example 2: Integration of the hypoxia-specific gene expression cassette into the dadX gene locus on the chromosome of the SWT004 strain (destroying its function)

[0398] 1. Pick a single clone of the alr gene-knockout attenuated bacteria SWT004 and inoculate it into 5 ml of fresh LB liquid culture medium. Place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0399] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue to culture for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0400] 3. Wash the bacteria 3 times with sterilized purified water.

[0401] 4. Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette. Mix primers SWTO78 and SWTO79 with the hypoxia-specific gene expression cassette plasmids pOL1001 and pOL1005, and a high-fidelity PCR amplification enzyme system and place them in a PCR amplification device.

[0402] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 120 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0403] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0404] 5. Mix the recovered bacteria with 100 ng of PCR product and electroporate at a voltage of 1.8 kV.

[0405] 6. The electroporated cells were spread on plates containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine and cultured overnight in a 32°C constant temperature incubator until single clones were grown. The resulting strains were SWT1001 and SWT1005.

[0406] 7. Inoculate the single clone into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours to allow the CRE enzyme to act and eliminate the chloramphenicol resistance gene.

[0407] 8. Using primers SWTO61, 20; SWTO75, 20, clones were identified by colony PCR. The identification results of strain SWT1001 (yhbU-S+alr+cyoA-S combination) and strain SWT1005 (yhbU-S+alr+ydcI-S combination) are shown in Figure 8.

[0408] Example 3: Construction of a hypoxia-specific gene expression cassette with dadX gene as an essential gene for survival

[0409] Since alanine racemase has two isoenzyme genes in the genomes of bacteria such as Salmonella and Escherichia coli, namely the alr gene and the dadX gene, to verify the effect, the essential genes for survival of some of the hypoxia-specific gene expression cassettes were replaced with the corresponding homologous genes dadX genes pOL2009 and pOL2013.

[0410] 1. Construction of dadX gene knockout attenuated bacteria (SWT007)

[0411] (1) A single clone of SWT003 was selected and inoculated into 5 ml of LB liquid culture medium, and then placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0412] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0413] (3) Wash the cells three times with sterilized purified water.

[0414] (4) Preparation of PCR products of SWTO78 and SWTO79.

[0415] Primers SWTO78 and SWTO79 were mixed with plasmid pSWT002 (same as above, plasmid description), and high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0416] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0417] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0418] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO78 and SWTO79 and electroporated at a voltage of 1.8 kV.

[0419] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0420] (7) Positive clones were identified by colony PCR, and SWTO59, SWTO5, SWTO6, and SWTO87 were used to identify the insertion of the chloramphenicol resistance gene, as shown in Figure 9.

[0421] (8) The positive single clone was inoculated into 5 ml of LB medium and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0422] 2. Construction of defective attenuated bacteria containing a hypoxia-specific gene expression cassette and the essential survival gene being the Salmonella dadX gene

[0423] (1) A single clone of the dadX gene-knockout attenuated bacteria SWT007 was selected and inoculated into 5 ml of LB liquid culture medium, and then placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0424] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0425] (3) Wash the cells three times with sterilized purified water.

[0426] (4) Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette. Mix primers SWTO85 and SWTO86 with the hypoxia-specific gene expression cassette plasmids pOL2009 and pOL2013, and a high-fidelity PCR amplification enzyme system and place them in a PCR amplification device.

[0427] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 120 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0428] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0429] (5) The recovered bacteria were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV.

[0430] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine and cultured overnight in a 32°C constant temperature incubator until a single clone colony was grown.

[0431] (7) The positive single clone was inoculated into 5 ml of LB medium and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0432] (8) Using the corresponding primers SWTO98, 22; SWTO99, 22, clones were identified by colony PCR. The identification results of strain SWT2013 (ynfK-E + dadX + ydcI-S) and strain SWT2009 (ynfK-E + dadX + cyoA-S) are shown in Figure 10.

[0433] Example 4: Verification of oxygen adaptability of Salmonella strains SWT1001, SWT1005, SWT2009, and SWT2013 containing a hypoxia-specific gene expression cassette

[0434] 1. Pick a single clone of strains SWT1001, SWT1005, SWT2009, and SWT2013 containing the hypoxia-specific expression cassette and inoculate it into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine. Place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0435] 2. After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0436] 3. Calculate the volume of bacterial solution per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0437] 4. Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, mark it as "1" and repeat three times, mark it as "a", "b", and "c".

[0438] 5. After 10-fold serial dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0439] 6. Repeat this 10-fold gradient dilution until you reach the mark "8".

[0440] 7. One plate was cultured at 37°C in an anaerobic environment, and the other plate was cultured at 37°C in an atmospheric environment (21% oxygen concentration).

[0441] The results of the oxygen adaptability verification test of the strains containing the hypoxia-specific expression cassette are shown in Figures 11 to 13. The results show that the Salmonella strains SWT1001, SWT1005, SWT2009, and SWT2013 containing the hypoxia-specific gene expression cassette exhibited hypoxia regulation capabilities.

[0442] Example 5: Oxygen Concentration Simulation of a Strain Library Containing Hypoxia-Specific Gene Expression Cassettes

[0443] By using anaerobic gas production bags to consume the oxygen in the sealed culture tank, and using an oxygen meter to measure the oxygen concentration in the sealed culture tank, the oxygen concentration in the sealed culture tank can be fixed within a specific concentration range after using the anaerobic gas production bags for a certain period of time. The specific method is as follows:

[0444] The anaerobic gas production bag (brand: Japan Mitsubishi, product number: D-119) was placed in a 7.0 L sealed culture tank (brand: Japan Mitsubishi, product number: D-112) and an oxygen meter (brand: Meicheng Electrochemical, product number: OX-100A) was placed in the tank.

[0445] The above anaerobic gas production bag combination was used to verify the growth of the genetically modified strain under an oxygen concentration below 0.8%.

[0446] Since the pathological hypoxic zone of a tumor is an oxygen concentration below 1%, the present invention intends to demonstrate that when the oxygen concentration is below 1% (or close to 1%), Salmonella transformed with the hypoxia-specific gene expression cassette can still grow normally.

[0447] Oxygen concentration control is accomplished by anaerobic gas production bags. After one hour of anaerobic gas production, the oxygen meter reading shows an oxygen concentration of 0.8-1%. Therefore, this experiment simulates the hypoxic environment within the tumor and verifies the growth of Salmonella on a petri dish after one hour of anaerobic gas production bags in a sealed culture jar. This also simulates the growth of Salmonella strains containing a hypoxia-specific gene expression cassette in the hypoxic environment within the tumor.

[0448] The operating procedures of the spot plate experiment are as follows:

[0449] (1) A single clone of the strain containing the hypoxia-specific expression cassette was selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0450] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0451] (3) Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0452] (4) Take 10 μl of the above bacterial solution and spot it on an LB plate containing D-alanine for culture, mark it as "1" and repeat three times, mark it as "a", "b", and "c".

[0453] (5) At the same time, take 10 μl of the above bacterial solution and spot it on another LB plate without D-alanine for culture, mark it as "1", and spot three replicates, mark them as "a", "b", and "c".

[0454] (6) After 10-fold serial dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as “2”.

[0455] (7) Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0456] (8) The two plates were placed in a 0.8% oxygen environment at 37°C and cultured.

[0457] As shown in Figure 14 , the present invention compared the growth of different strains (Figure 14 (A) SWT1001, Figure 14 (B) STW1005, Figure 14 (C) SWT2009, and Figure 14 (D) SWT2013) on LB plates without D-alanine to determine the growth performance of each strain at oxygen concentrations below 0.8%. Because the culture medium without D-alanine requires the corresponding Salmonella strains to rely on their own oxygen regulation systems for normal growth, the results confirmed that strains containing the hypoxia-specific expression cassette, which can grow normally in an anaerobic environment, can also grow normally at oxygen concentrations below 0.8%.

[0458] Example 6: Construction of Salmonella strains (SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, SWT2013-T7P) with T7 RNA polymerase homologous replacement of the asd gene site

[0459] (1) Construction of lacUV5-controlled T7 RNA polymerase constitutive expression plasmid pPRO005

[0460] To construct a constitutively expressed T7 RNA polymerase, the vector pPRO005 was constructed. The process, as shown in Figure 15 , involves placing the constitutively expressed promoter lacUV5 upstream of the RBS sequence (SEQ ID No. 35) and T7 RNA polymerase (SEQ ID No. 4). Furthermore, the chloramphenicol resistance gene cm (SEQ ID No. 37) flanked by loxP sequences was added upstream of lacUV5. This facilitates recombination onto the Salmonella chromosome and facilitates screening.

[0461] 1. Plasmid pSWT007 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with SpeI and NotI to recover an approximately 1100 bp fragment containing the chloramphenicol resistance gene cm flanked by loxP sequences.

[0462] 2. Direct annealing of primers SWTO100 and SWTO101 to generate NotI and HindIII cuts and recover a fragment of approximately 70 bp, including the promoter lacUV5;

[0463] 3. Plasmid pPRO004 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with HindIII and XhoI to recover a 2690 bp fragment including the T7 RNA polymerase gene;

[0464] 4. Cut plasmid pSWT003 (plasmid backbone) with SpeI and XhoI to recover a fragment of approximately 3000 bp;

[0465] 5. The above five fragments were ligated by T4 DNA ligase, transformed into DH10B competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and spread on ampicillin and chloramphenicol double-resistant LB plates, and single clones were screened and named pPRO005.

[0466] 6. Plasmid pPRO005 was extracted, and 100 ng was mixed with NotI and XhoI endonuclease system, incubated at 37°C for 1 hour, and identified by agarose gel electrophoresis, as shown in Figure 16.

[0467] (ii) Recombinant lacUV5 controls the T7 RNA polymerase constitutive expression cassette to the asd gene position on the Salmonella chromosome

[0468] 1. Pick single clones from SWT1001, SWT1005, SWT2009, and SWT2013, respectively, and inoculate them into 5 ml LB liquid culture medium containing 100 μg / ml D-alanine. Place the culture medium in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0469] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue to culture for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place it on ice for 1 hour.

[0470] 3. Wash the bacteria 3 times with sterilized purified water.

[0471] 4. Prepare PCR products of SWTO102 and SWTO103.

[0472] Primers SWTO102 and SWTO103 were mixed with plasmid pPRO005 and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0473] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0474] The amplified PCR products were subjected to agarose gel electrophoresis to verify the PCR amplification products of SWTO102 and SWTO103. The verification results are shown in FIG17A .

[0475] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0476] 5. The recovered bacteria were mixed with 100 ng of PCR products of SWTO102 and SWTO103 and electroporated at a voltage of 1.8 kV.

[0477] 6. Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol, 100 μg / ml D-alanine, and 100 μg / ml DAP (diaminopimelic acid), and culture in a 32°C incubator overnight until a single clone colony grows.

[0478] 7. Inoculate the single clone into 5 ml of LB medium containing 100 μg / ml DAP and 100 μg / ml D-alanine, and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0479] 8. Identify positive clones by colony PCR, using SWTO104, SWTO106, SWTO105, and SWTO107 for identification.

[0480] The target fragment PCR amplification products of strains SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P were verified by agarose gel electrophoresis. The verification results are shown in Figure 17B.

[0481] Example 7: Construction of eukaryotic drug mRNA / DNA mixed dual delivery cytoplasmic green fluorescent protein (EGFP) expression vector pEU010 and control vector pEU011

[0482] 1. Construction of the control vector pEU011 for single cytoplasmic delivery of eukaryotic drug mRNA

[0483] In this example, to verify the system of single cytoplasmic delivery of eukaryotic drug mRNA, a control plasmid vector pEU011 carrying eukaryotic expression of EGFP as an example, without self-enhancing regulation and antibiotic resistance selection, was constructed, as shown in Figure 18A.

[0484] The pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were used as balanced lethal control mechanisms, and a T7 promoter containing a double phage source was used to control the expression of the drug protein (in the plasmid pEU011, this element was replaced by EGFP, SEQ ID No. 17) and the membrane-breaking protein gene LLO (SEQ ID No. 13), respectively.

[0485] The specific steps are as follows:

[0486] (1) Plasmid pPRO006 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with XhoI and NotI to recover an approximately 820 bp fragment containing the pUC replicon (SEQ ID No. 36);

[0487] (2) Plasmid pEU001 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with SpeI and XhoI to recover a 2831 bp fragment containing the Salmonella asd gene and its regulatory region, the LLO gene, and the RBS site (SEQ ID No. 15);

[0488] (3) Plasmid pEU002 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NotI and NcoI to recover a fragment of approximately 654 bp containing the first IRES sequence under the control of the T7 promoter and the kozak sequence (SEQ ID No. 16);

[0489] (4) Plasmid pEGFP-N1 (purchased from Biowind) was digested with NcoI and XbaI to recover a fragment of approximately 731 bp containing EGFP (SEQ ID No. 17);

[0490] (5) Plasmid pEU002 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with XbaI and SpeI to recover a fragment of approximately 281 bp containing the 3′-UTR region, a 50 bp polyA sequence, a T7 terminator, and a second T7 promoter (SEQ ID No. 18);

[0491] (6) The above five fragments were ligated using T4 DNA ligase, transformed into SWT1005-T7P competent cells, and coated on 100 μg / ml D-alanine LB plates. A single clone was selected and named SWT5015C.

[0492] (7) A single clone was picked and amplified using primers SWTO129 and SWTO130; SWTO131 and SWTO132; and identified by agarose gel electrophoresis. As shown in FIG. 19 , the resulting plasmid was pEU011.

[0493] 2. Construction of the eukaryotic drug mRNA / DNA dual delivery cytoplasmic expression vector pEU010

[0494] In this example, to verify the system of dual delivery of eukaryotic drug mRNA / DNA to the cytoplasm, a plasmid vector pEU010 carrying eukaryotic expression of EGFP as an example and without antibiotic resistance selection was constructed, as shown in FIG20 .

[0495] The pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were used as balanced lethal control mechanisms, and a T7 promoter containing a dual phage source was used to control the expression of the drug protein (in plasmid pEU010, this element was replaced by EGFP, SEQ ID No. 17), the transcription of T7 RNA polymerase (SEQ ID No. 20), and the membrane-breaking protein gene LLO (SEQ ID No. 13), respectively.

[0496] The specific steps are as follows:

[0497] (1) Plasmid pEU011 was digested with XbaI and SacI to recover a fragment of approximately 5322 bp;

[0498] (2) Plasmid pEU002 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was amplified using primers SWTO133 and SWTO134, and a fragment of approximately 613 bp containing the IRES sequence was recovered. The fragment was then cloned using XbaI and EcoRI, and this fragment was used as the second IRES sequence (SEQ ID No. 19);

[0499] (3) Plasmid pPRO004 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was amplified using primers SWTO135 and SWTO136, and a fragment of approximately 2670 bp containing the T7 RNA polymerase sequence was recovered. This fragment was then cloned using EcoRI and SacI, and this fragment served as the T7 RNA polymerase sequence driven by the second IRES sequence (SEQ ID No. 20);

[0500] (4) The three fragments were ligated using T4 DNA ligase, transformed into competent cells of SWT1005-T7P, and plated on LB plates containing 100 μg / ml D-alanine. A single clone was selected and named SWT5015.

[0501] (5) A single clone was picked and amplified using primers SWTO137 and SWTO134; SWTO137 and SWTO138; and identified by agarose gel electrophoresis. As shown in FIG. 21 , the resulting plasmid was pEU010, with the sequence being SEQ ID No. 21.

[0502] 3. Construction of cytoplasmic expression vector of patent US10987432B2 as a control

[0503] The structure of the cytoplasmic expression vector of patent US10987432B2 is shown in FIG22 , and the plasmid pIKDE-EGFP (SEQ ID No. 22) was synthesized based on the sequence provided in the patent and synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0504] Example 8: Comparison of expression intensity of expression vector pEU010 and control vectors pEU011 and pIKDE-EGFP in BSR-T7 / 5 golden hamster kidney cells stably expressing T7 polymerase

[0505] In this example, in order to verify the difference in EGFP expression intensity between the expression vector pEU010 for the mixed dual delivery of eukaryotic drug mRNA / DNA to the cytoplasm, the control vector pEU011 for the single delivery of eukaryotic drug mRNA to the cytoplasm, and the cytoplasmic expression vector pIKDE-EGFP of patent US10987432B2, the three plasmids were transfected into BSR-T7 / 5 golden hamster kidney cells (purchased from Shanghai Qiansi Biotechnology Co., Ltd.) stably expressing T7 RNA polymerase. Since BSR-T7 / 5 carries its own T7 RNA polymerase, the gene on the plasmid can be transcribed directly in the cytoplasm. The advantages and disadvantages of different systems were verified by comparing the strength of the green fluorescence expression of EGFP.

[0506] The specific steps are as follows:

[0507] 1. Culture BSR-T7 / 5 cells. Culture cells in 24-well plates using DMEM (purchased from Shanghai Source Biotechnology Co., Ltd., Catalog No. L110KJ) supplemented with 10% FBS serum (purchased from US Everbright, Catalog No. H9043). One day before transfection (18-24 hours), seed approximately 100,000-300,000 cells per well in the 24-well plate and culture until the cell density reaches approximately 70-80% on the next day. Two hours before transfection, replace each well of the 24-well plate containing cells with 1 ml of fresh complete culture medium containing serum and penicillin-streptomycin antibiotics (purchased from Shanghai Source Biotechnology Co., Ltd., Catalog No. S110JV).

[0508] 2. Take a clean centrifuge tube and add 25 μl Opti-MEM (purchased from Gibco, product number 31985-070) to each well of the cells in the 24-well plate to be transfected. Add 500 ng of plasmids pEU10, pEU11, pIKDE-EGFP, DNA (0.5-5 μg / μl) to each well and mix them by gently pipetting with a gun. Then add 0.8 μl Lipo8000 TM Gently pipette and mix the transfection reagent (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., Cat. No. C0533). Add the DNA and Lipo8000 mixture evenly to each well according to the above dosage and continue incubation.

[0509] 3. After culturing for about 48 hours, the expression of green fluorescence can be observed using a fluorescence microscope and the proportion of fluorescent cells can be analyzed by flow cytometry.

[0510] The fluorescence microscopy results are shown in Figure 23. Figure 23A is a BSR-T7 / 5 cell control, with the upper part showing white light and the lower part showing a fluorescence image, and the result shows no fluorescence signal; Figure 23B shows BSR-T7 / 5 cells transfected with pEU010, and a large number of cells with fluorescence signals were found; Figure 23C shows BSR-T7 / 5 cells transfected with pEU011, and cells with fluorescence signals were found, but the number was significantly less than that in Figure 23B; Figure 23D shows BSR-T7 / 5 cells transfected with pIKDE-EGFP, and cells with fluorescence signals were found, but the number was significantly less than that in Figure 23B.

[0511] Flow cytometric analysis results are shown in Figure 24 . Green fluorescent protein-positive cells were detected in the FITC channel, with the P3 gate set to green fluorescent protein-positive cells. Figure 24A shows BSR-T7 / 5 cells transfected with pEU010, with a positive cell ratio of 44.1%; Figure 24B shows BSR-T7 / 5 cells transfected with pEU011, with a positive cell ratio of 35.7%; and Figure 24C shows BSR-T7 / 5 cells transfected with pIKDE-EGFP, with a positive cell ratio of 34.8%.

[0512] According to the above two experiments, the plasmids of these three systems can achieve the synthesis of target proteins in cells with a T7 RNA polymerase expression background. The eukaryotic drug mRNA / DNA mixed dual delivery cytoplasmic expression vector pEU010 has obvious advantages compared with the other two control vectors.

[0513] Example 9: Construction of strain SWT5025 carrying the control vector pIKDE-EGFP as a control strain according to patent US10987432B2

[0514] 1. Construction of YB1-like Salmonella (SWT005)

[0515] In order to restore the strain carrying the control vector pIKDE-EGFP of patent US10987432B2, the first step is to construct the chassis strain of the original YB1.

[0516] The YB1-like Salmonella strain includes aroA gene deficiency and an oxygen regulation system constructed by the forward low-oxygen promoter pepT, the essential gene asd, and the reverse high-oxygen promoter sodA.

[0517] (1) Construction of plasmid pSWT004

[0518] ① The pSWT004 plasmid is shown in Figure 25, which carries the chloramphenicol resistance gene cm (SEQ ID No.39) with loxp sequences on both sides, the pepT promoter (SEQ ID No.41), the asd gene (SEQ ID No.42), and the reverse sodA promoter (SEQ ID No.43).

[0519] ② Plasmid pSWT002 was digested with NotI and the 1130 bp fragment was recovered. Strain SWT001 was amplified using primers SWTO11 and SWTO12, and the product was double-digested with NotI and HindIII and recovered.

[0520] Strain SWT001 was amplified using primers SWTO13 and SWTO14, and the product was double-cut with XhoI and HindIII and recovered.

[0521] Primers SWTO15 and SWTO16 were directly annealed to generate XhoI and PstI nicks, and then recovered.

[0522] ③ The plasmid pSWT003 (plasmid backbone) was double-cut with NotI and PstI, and the five fragments were connected by T4 DNA ligase. The plates were coated with ampicillin and chloramphenicol double-resistant LB plates, and single clones were screened and named pSWT004 (YB1-like construction plasmid, including cm-pepT-asd-sodA).

[0523] ④ Extract plasmid pSWT004, take 100 ng and mix with SacI and KpnI endonuclease system, incubate at 37°C for 1 hour, and identify by agarose gel electrophoresis, as shown in Figure 26.

[0524] (2) Construction of recombinant YB1-like Salmonella SWT005 (YB1-like)

[0525] Plasmid pSWT004 was amplified using primers SWTO17 and SWTO18, recovered, and recombined into SWT003 to construct YB1-like Salmonella SWT005.

[0526] ① Pick a single clone of SWT003 and inoculate it into 5 ml of LB liquid culture medium, and place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0527] ②Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue to culture for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0528] ③ Wash the bacteria 3 times with sterilized purified water and set aside.

[0529] ④ pSWT004 was amplified using primers SWTO17 and SWTO18, and the recovered PCR products were purified and recovered. The PCR amplification products of the cm-pepT-asd-sodA fragments SWTO17 and SWTO18 were verified by agarose gel electrophoresis. The verification results are shown in Figure 27. The DNA concentration and purity were determined by nanodrop.

[0530] ⑤ Mix the recovered bacteria with 100 ng of PCR product and electroporate at a voltage of 1.8 kV.

[0531] ⑥ Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml DAP (diaminopimelic acid), and culture them in a 32°C constant temperature incubator overnight until a single clone colony grows.

[0532] ⑦ Positive clones were identified by colony PCR. SWTO93, SWTO5, SWTO94, and SWTO95 were used to identify the insertion of the chloramphenicol resistance gene. The results of verification of the PCR amplification products of Salmonella SWT005 by agarose gel electrophoresis (SWTO93 and SWTO5, SWTO94 and SWTO95) are shown in Figure 28.

[0533] (3) Verification of oxygen adaptability of YB1-like Salmonella SWT005

[0534] ① Pick a single clone of YB1-like Salmonella SWT005 and inoculate it into 5 ml of LB liquid culture medium containing 100 μg / ml DAP (diaminopimelic acid), and place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0535] ② After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0536] ③ Calculate the volume of bacterial solution (μl) per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0537] ④ Take 10 μl of the above bacterial solution and spot it on two LB plates without DAP for culture, mark it as "1", and repeat three times, mark it as "a", "b", and "c".

[0538] ⑤ After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marked as "2".

[0539] ⑥ Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0540] ⑦ One of the plates was placed in an anaerobic jar at 37°C. The operation process was to place the anaerobic gas-generating bag (brand: Japan Mitsubishi, product number: D-119) in a 7.0L sealed culture jar (brand: Japan Mitsubishi, product number: D-112) and cultured in an anaerobic environment (oxygen concentration less than 0.01%) for 24 hours. The other plate was placed in an atmospheric environment at 37°C (21% oxygen concentration) and cultured for 24 hours.

[0541] The results of the oxygen adaptability verification test of YB1-like Salmonella SWT005 are shown in Figure 29. From the oxygen adaptability verification data of YB1-like Salmonella SWT005, it can be concluded that YB1-like Salmonella SWT005 can grow normally in the absence of oxygen on the LB plate without DAP (as shown in Figure 29 (A)), and can grow normally in the presence of oxygen (atmospheric environment) at a concentration of 1 (10 -2 OD), concentration 2(10 -3 OD), concentration 3(10 -4 OD), concentration 4(10 -5 OD) showed obvious growth of the strains (as shown in Figure 29 (B)).

[0542] Therefore, it can be seen that YB1-like Salmonella SWT005 achieves regulation of strain growth in aerobic environment, which is consistent with previous reports on YB1.

[0543] 2. Construction of strain SWT008 with T7 RNA polymerase expression system controlled by placUV5 promoter introduced at gmd position on chromosome on YB1-like Salmonella (SWT005) chassis

[0544] Since the asd gene position of the SWT005 strain was occupied by a hypoxia-regulated gene cassette, T7 RNA polymerase controlled by the placUV5 promoter was integrated into the gmd position with reference to patent US10987432B2.

[0545] The specific steps are as follows:

[0546] (1) A single clone of SWT005 was selected and inoculated into 5 ml LB liquid culture medium containing 100 μg / ml DAP, and then placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0547] (2) Inoculate the culture into fresh 100 μg / ml DAP LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600=0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place it on ice for 1 hour.

[0548] (3) Wash the cells three times with sterilized purified water.

[0549] (4) Preparation of PCR products of SWTO139 and SWTO140.

[0550] Primers SWTO139 and SWTO140 were mixed with plasmid pPRO005 and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0551] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0552] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0553] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO139 and SWTO140 and electroporated at a voltage of 1.8 kV.

[0554] (6) The electroporated cells were spread on plates containing 25 μg / ml chloramphenicol and 100 μg / ml DAP and cultured overnight in a 32°C constant temperature incubator until a single clone colony was grown.

[0555] (7) A single clone was inoculated into 5 ml of LB medium containing 100 μg / ml DAP and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The chloramphenicol resistance gene was deleted through the action of CRE enzyme to generate strain SWT008.

[0556] (8) Positive clones were identified by colony PCR using SWTO141, SWTO106, SWTO142, and SWTO107.

[0557] The target fragment PCR amplification product of strain SWT008 was verified by agarose gel electrophoresis, and the verification results are shown in Figure 30.

[0558] 3. Construction of strain SWT5025 carrying the control vector pIKDE-EGFP

[0559] According to US Patent No. 10987432B2, to ensure stable plasmid carriage in Salmonella, pIKDE-EGFP contains an essential gene, the infA gene. The infA gene is a protein translation initiation factor and is crucial for viability. Therefore, the construction of strain SWT5025 requires transferring the plasmid pIKDE-EGFP into strain SWT008 and subsequently deleting the infA gene on the chromosome to ensure the functioning of the balanced lethal system.

[0560] In addition, in patent US10987432B2, the expression of the LLO gene is achieved on the chromosome by using the promoter of the sseA gene on the Salmonella pathogenicity island II (SEQ ID No. 40). The sseA promoter is a strong promoter that can be induced to express after the bacteria invade the cell. Therefore, in the present invention, vector pPRO013 was constructed, which contains the sseA promoter-driven LLO gene expression, and also includes two oriented loxP sequences on both sides and a chloramphenicol resistance gene in the middle. This fragment was then amplified by PCR and recombined into the infA position on the SWT008 chromosome to generate strain SWT5025.

[0561] The specific steps are as follows:

[0562] (1) The plasmid pIKDE-EGFP was transformed into competent cells of SWT008 and spread on LB plates containing 100 μg / ml DAP and 100 μg / ml ampicillin sodium. A single clone was selected and named SWT009.

[0563] (2) A single clone of SWT009 was picked and inoculated into 5 ml LB liquid culture medium containing 100 μg / ml DAP and 100 μg / ml ampicillin sodium, and cultured in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0564] (3) Inoculate the culture into fresh 100 μg / ml DAP LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place it on ice for 1 hour.

[0565] (4) Wash the cells three times with sterilized purified water.

[0566] (5) Prepare the PCR amplification products of pPRO013 of SWTO143 and SWTO144.

[0567] The pPRO013 plasmid was constructed as shown in FIG31 .

[0568] ① Plasmid pPRO009 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NdeI and PstI to recover a fragment of approximately 1600 bp containing the codon-optimized Salmonella gene LLO;

[0569] ② Plasmid pPRO011 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NotI and NdeI to recover a fragment of approximately 425 bp containing the sseA promoter and RBS site sequence. This fragment served as the LLO promoter.

[0570] ③ The plasmid pSWT002 was digested with NotI and the 1130 bp fragment was recovered;

[0571] ④ The plasmid pSWT003 (plasmid backbone) was double-cut with NotI and PstI, and the four fragments were connected by T4 DNA ligase. The plates were coated with ampicillin and chloramphenicol double-resistant LB plates, and a single clone was selected and named pPRO013.

[0572] (6) Mix primers SWTO143 and SWTO144 with plasmid pPRO013 and high-fidelity PCR amplification enzyme system and place them in a PCR amplification device.

[0573] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0574] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0575] (7) The recovered cells were mixed with 100 ng of PCR products of SWTO143 and SWTO144 and electroporated at a voltage of 1.8 kV.

[0576] (8) The electroporated cells were spread on plates containing 25 μg / ml chloramphenicol and 100 μg / ml DAP and cultured overnight in a 32°C incubator until a single clone colony was grown.

[0577] (9) A single clone was inoculated into 5 ml of LB medium containing 100 μg / ml DAP and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The chloramphenicol resistance gene was deleted through the action of CRE enzyme to generate strain SWT5025.

[0578] (10) Positive clones were identified by colony PCR using SWTO145, SWTO5, SWTO146, and SWTO116.

[0579] The target fragment PCR amplification product of strain SWT5025 was verified by agarose gel electrophoresis, and the verification results are shown in Figure 32.

[0580] Example 10: Comparison of the performance and cell presentation efficiency of strains SWT5015, SWT5015C, and SWT5025

[0581] In this example, the performance of strain SWT5015 expressing the eukaryotic drug mRNA / DNA dual-delivery cytoplasmic green fluorescent protein (EGFP) expression vector pEU010, strain SWT5015C expressing the control vector pEU011, and strain SWT5025 expressing pIKDE-EGFP were compared in terms of strain stability, membrane permeabilization protein expression rate, and EGFP expression efficiency. Table 2 illustrates the characteristics of these three systems.

[0582] Table 2 Comparison of characteristics of strains SWT5015, SWT5015C and SWT5025

[0583] 1. Comparison of the stability of strains SWT5015, SWT5015C, and SWT5025

[0584] Both strains SWT5015 and SWT5025 utilize a self-enhanced regulatory model using eukaryotically expressed T7 RNA polymerase on a plasmid. Although self-enhanced eukaryotic T7 RNA polymerase has low expression in the host bacteria, the presence of constitutively expressed T7 RNA polymerase on the chromosome allows this self-enhanced model to induce cyclical self-enhanced transcription in the host bacteria, leading to bacterial death due to internal resource depletion.

[0585] In this example, by using SWT1005 as a control, the growth and survival status of three strains, SWT5015, SWT5015C and SWT5025, in a normal culture medium environment were compared to determine the stability of the strains.

[0586] The specific steps are as follows:

[0587] (1) Single clones of SWT1005, SWT5015, and SWT5015C were respectively inoculated into 5 ml LB liquid medium containing 100 μg / ml D-alanine; single clones of SWT5025 were inoculated into 5 ml LB liquid medium containing 100 μg / ml DAP and 100 μg / ml ampicillin sodium, and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0588] (2) SWT1005, SWT5015, SWT5015C, and SWT5025 cultures were inoculated into fresh liquid culture medium at a ratio of 1:1000, and transferred to a 96-well cell culture plate. The cells were cultured in a constant temperature shaker at 37°C and 220 rpm for 24 hours, and the bacterial density (OD) was measured every hour using a microplate reader. 600 Once and record the growth status, see Figure 33A.

[0589] (3) After the culture is complete, dilute the strain 10-fold and measure the absorbance (OD) at 600 nm. Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0590] (4) Take 10 μl of the above-mentioned SWT1005, SWT5015, and SWT5015C bacterial solutions and spot them on an LB plate containing D-alanine for culture. Take 10 μl of the SWT5025 bacterial solution and spot them on an LB plate containing DAP for culture, marked as "1". Repeat three times, marked as "a", "b", and "c". After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above-mentioned culture medium, marked as "2". Repeat this 10-fold gradient dilution until the mark "8" is reached. The growth status is shown in Figure 33B.

[0591] The results showed that SWT5015 and SWT5015C showed no difference in growth rate in liquid culture or in plate counts compared to the control strain SWT1005. This demonstrated that the self-enhancing regulation of SWT5015 had no impact on strain stability. However, SWT5025 exhibited a significantly slower growth rate in liquid culture, and flocculent precipitates were observed in the culture medium during the culture process, representing dead bacteria. This resulted in lower overall OD readings in the later stages of the culture than those of SWT5015, SWT5015C, and the control strain SWT1005.

[0592] Furthermore, spot plate counts revealed that at the same OD, the number of viable bacteria in SWT5025 was 100-fold lower than in other strains, representing only 1% of the viable counts in other strains. This suggests that SWT5025's self-regulatory behavior significantly impacts bacterial survival.

[0593] 2. Comparison of EGFP mRNA transcription in SWT5015, SWT5015C, and SWT5025 strains

[0594] Since SWT5015, SWT5015C, and SWT5025 strains can present mRNA and constitutively express T7 RNA polymerase, the target protein, eukaryotic expression of EGFP, is present in bacteria. Therefore, in this example, the levels of eukaryotic expression of EGFP mRNA in these three strains were identified by reverse transcription-qPCR, using SWT1005 as a control.

[0595] The specific steps are as follows:

[0596] (1) Single clones of SWT1005, SWT5015, and SWT5015C were respectively inoculated into 5 ml LB liquid medium containing 100 μg / ml D-alanine; single clones of SWT5025 were inoculated into 5 ml LB liquid medium containing 100 μg / ml DAP and 100 μg / ml ampicillin sodium, and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0597] (2) Transfer the cultured strains into centrifuge tubes containing 1 ml of fresh corresponding culture medium at a ratio of 1:100, and culture at 32°C, 200 rpm for 4-6 hours until the OD 600 Between 0.5-1.

[0598] (3) Centrifuge the bacterial suspension of each strain at 4°C, 12,000 rpm for 2 minutes, and carefully remove all the supernatant.

[0599] (4) Resuspend the cells thoroughly (using a pipette) in 100 μl of TE buffer containing lysozyme (purchased from Shanghai Biotech Biotechnology Co., Ltd., catalog number ST206) and incubate at room temperature for 5 minutes.

[0600] (5) Add 300 μl of Lysis buffer (GeneJET RNA purification kit, purchased from Thermo, catalog number K0731) to each tube and vortex to mix.

[0601] (6) Add 180 μl of anhydrous ethanol to each tube and mix thoroughly by pipetting. Transfer all the liquid in the centrifuge tube to an RNA purification column (GeneJET RNA Purification Kit, purchased from Thermo, Cat. No. K0731), centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid. Transfer the adsorption column to a new 2 ml collection tube.

[0602] (7) Add 700 μl of wash buffer 1 (GeneJET RNA purification kit, purchased from Thermo, product number K0731) to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0603] (8) Add 600 μl of wash buffer 2 (GeneJET RNA purification kit, purchased from Thermo, product number K0731) to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0604] (9) Transfer the adsorption column to a new 1.5 ml centrifuge tube, add 50 μl of DEPC water (purchased from Shanghai Biyuntian Biotechnology Co., Ltd., product number R0022), let it stand at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, and discard the adsorption column.

[0605] (10) The RNA tube was placed on ice, and its concentration and purity were detected by Nanodrop. 500 ng was then taken and run on 1% electrophoresis to detect its integrity. The identification results are shown in FIG34A.

[0606] (11) Remove residual DNA from RNA. Pipette 1 μl of extracted RNA, add 1 μl of DNaseI (purchased from Thermo, catalog number EN0521), 1 μl of DNaseI buffer, and make up to 10 μl with DEPC water. Incubate at 37°C for 30 minutes and inactivate at 65°C for 10 minutes.

[0607] (12) Pipette 1 μl of RNA from the previous step, mix it with the corresponding primers SWTO147 and SWTO148, Luna Universal qPCR Master Mix (purchased from NEB, catalog number M3003S) and amplification enzyme system, and then place the mixture into the fluorescent PCR amplification device.

[0608] The amplification program was 95°C for 60 seconds, 95°C for 15 seconds, and 60°C for 30 seconds, for 40 cycles.

[0609] (13) Reverse transcription was not performed in this step to eliminate the possibility of residual DNA interfering with the experiment. The results are shown in Figure 34B.

[0610] (14) Pipette 1 μl of RNA from step (11), mix it into the corresponding primers SWTO147 and SWTO148 Luna Universal One-step RT-qPCR kit (purchased from NEB, product number E3005S) amplification enzyme system, and then place the mixture into the fluorescent PCR amplification device.

[0611] The amplification program was 55°C for 10 minutes; 95°C for 60 seconds; 95°C for 15 seconds, 60°C for 30 seconds, and 40 cycles. The purpose was to first convert mRNA into cDNA by reverse transcription and then amplify the target gene. The results are shown in Figure 34B.

[0612] The above experimental results showed that the SWT5015, SWT5015C and SWT5025 strains all contained the mRNA of the target gene EGFP, and there was no significant difference in the levels of their mRNA.

[0613] 3. Comparison of expression efficiency of T7 RNA polymerase and membrane permeabilization protein LLO in strains SWT5015, SWT5015C, and SWT5025

[0614] The T7 RNA polymerase of strains SWT5015, SWT5015C and SWT5025 are all controlled by the constitutive promoter lacUV5 and expressed on the chromosome. However, the expression regulation of LLO of strains SWT5015 and SWT5015C is controlled by the T7 promoter and is located on the plasmid of the expression vector. The advantage is that it can achieve synchronous expression with the mRNA of the target protein, and the storage of sufficient LLO protein in the bacteria can maximize the efficiency of the overall bacterial delivery. In SWT5025, it is controlled by the promoter (SEQ ID No.40) of the sseA gene on the Salmonella pathogenicity island II on the chromosome, and is induced to express after the bacteria invade the cell.

[0615] In this example, the expression of T7 RNA polymerase and LLO protein of SWT5015, SWT5015C and SWT5025 strains in ordinary culture medium and sseA promoter induction medium N-salts (containing low concentration of magnesium ions to simulate the post-cell invasion environment and induce sseA promoter expression; the ingredients are 5mM KCl, 7.5mM (NH4)2SO4, 0.5mM K2SO4, 100mM Bis-Tris / HCl (pH 7.0), 38mM glycerol, and 0.1% Casamino Acids with 30μM MgCl2 added) was verified.

[0616] The specific steps are as follows:

[0617] (1) Single clones of SWT5015 and SWT5015C were respectively inoculated into 5 ml LB liquid medium containing 100 μg / ml D-alanine; single clones of SWT5025 were inoculated into 5 ml LB liquid medium containing 100 μg / ml DAP and 100 μg / ml ampicillin sodium, and placed in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0618] (2) Take 1 ml of each bacterial strain and centrifuge at 12000 RPM for 1 minute. Remove the supernatant and recover the precipitate for later use.

[0619] (3) Take 1 ml of each strain culture medium, centrifuge at 4000 RPM for 5 minutes, remove the supernatant, and resuspend in 5 ml of freshly prepared induction medium N-salts. Add 100 μg / ml D-alanine to the culture medium of SWT5015 and SWT5015C; add 100 μg / ml DAP and 100 μg / ml ampicillin sodium to the culture medium of SWT5025. Place them in a constant temperature shaker at 37°C and 220 rpm for 24 hours.

[0620] (4) Take 5 ml of the bacterial solution after culturing in the induction medium N-salts for each strain, centrifuge at 12000 RPM for 1 minute, remove the supernatant, and recover the precipitate for later use.

[0621] (5) The samples in step (2) and step (4) were resuspended in lysis buffer (protein loading buffer, purchased from CST, product number 7722S), heated at 95°C for 10 minutes, centrifuged again at 12,000 rpm, and the supernatant was recovered for later use.

[0622] (6) 5 μl of the supernatant recovered in the previous step was taken and added to SDS-PAGE gel (SDS-PAGE gel preparation kit purchased from Sangon Biotechnology, product number C631100-0200), and electrophoresed at 120 V for 60 minutes.

[0623] (7) The SDS-PAGE gel after electrophoresis was transferred to a PVDF membrane (purchased from Roche, product number 03010040001) at a steady current of 250 mA for 30 minutes.

[0624] (8) The PVDF membrane was transferred to 5% skim milk powder and incubated at room temperature for 1 hour on a horizontal shaker at 80 rpm as a blocking method.

[0625] (9) The blocked PVDF membrane was incubated with anti-T7 RNA polymerase antibody (T7 RNA polymerase antibody, purchased from Creative Biomart, catalog number CABT-B8990) and anti-listeriolysin antibody (LLO identification antibody, purchased from abcam, catalog number ab200538) as primary antibodies diluted with 5% skim milk powder at 4°C for 18 hours (one antibody was used for each membrane).

[0626] (10) Dilute the secondary antibody with 5% skim milk powder, transfer the PVDF membrane after primary antibody incubation to the secondary antibody dilution solution, and incubate on a horizontal shaker at 80 rpm for 1 hour at room temperature.

[0627] (11) Wash the PVDF membrane after secondary antibody incubation with TBST Buffer (purchased from Sangon Biotechnology, product number C520009-0005) three times, 5 minutes each time. Add ECL developer (developer SignalFire) to the washed PVDF membrane. TM ECL Reagent (CST, Cat. No. 6883p3) was purchased and incubated in the dark for 1 min. After incubation, the PVDF membrane was placed on a chemiluminescence instrument for image acquisition.

[0628] Western blot analysis revealed that strains SWT5015, SWT5015C, and SWT5025 all expressed T7 RNA polymerase at high levels under both LB and N-salts culture conditions, as shown in Figure 35A. Western blot analysis revealed that strains SWT5015 and SWT5015C also expressed LLO at high levels under both LB and N-salts culture conditions, as shown in Figure 35B. SWT5025 only expressed LLO under N-salts culture conditions, as shown in Figure 35B. This temporal and spatial variation in LLO expression may affect the efficiency of mRNA / DNA presentation after bacterial invasion.

[0629] 4. Comparison of EGFP expression efficiency after SWT5015, SWT5015C, and SWT5025 invade cancer cells

[0630] In the above examples, the stability and T7 polymerase and LLO expression efficiencies of SWT5015, SWT5015C, and SWT5025 were verified. Although differences were found, further evaluation of drug delivery efficiency requires cell invasion assays.

[0631] In this example, SWT1005 was used as a negative control, and SWT5015, SWT5015C, and SWT5025 strains were co-cultured with CT26 cancer cells to analyze the efficiency differences from the perspective of fluorescent protein expression.

[0632] The specific steps are as follows:

[0633] (1) Single clones of SWT1005, SWT5015, and SWT5015C were respectively inoculated into 5 ml LB liquid medium containing 100 μg / ml D-alanine; single clones of SWT5025 were inoculated into 5 ml LB liquid medium containing 100 μg / ml DAP and 100 μg / ml ampicillin sodium, and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0634] (2) Take 1OD of culture and wash three times with PBS.

[0635] (3) Add 2.5×10 6 CT26 cancer cells were cultured in complete culture medium (DMEM basal medium + 10% fetal bovine serum + double antibody) in a 5% CO2 incubator at 37°C overnight.

[0636] (4) After counting the cells in two wells, the SWT1005, SWT5015, SWT5015C, and SWT5025 strains were added according to the cell number at an infection ratio of 200:1. The cells were placed in an anaerobic jar with an oxygen concentration below 0.8% and cultured for 2 hours to allow the bacteria to invade the cells. The cells were then carefully washed with an equal volume of PBS, and the supernatant was aspirated. DMEM medium containing 100 μg / mL gentamicin was added and cultured in an anaerobic jar with an oxygen concentration below 0.8% for another 24 hours. The cells were photographed under a fluorescence microscope as shown in Figure 36A.

[0637] (5) The invaded cells were digested with 0.25% trypsin (purchased from Shanghai Yuanpei Biotechnology Co., Ltd., catalog number S310JV), resuspended in 100 μl of PBS, transferred to a black flat-bottom 96-well plate, and placed in a microplate reader to read the green fluorescence signal at room temperature (emission wavelength 518 nm, absorption wavelength 518 nm, reading time fixed at 800 ms). The results are shown in Figure 36B.

[0638] As shown above, SWT1005, a negative control, showed no fluorescence signal when analyzed by a microplate reader. EGFP signals were detected in all strains SWT5015, SWT5015C, and SWT5025, with SWT5015 showing the strongest signal. Due to the relatively low efficiency of bacterial eukaryotic delivery systems, only the mRNA / DNA hybrid of SWT5015 was efficiently delivered to the cytoplasm, resulting in a clearly observable fluorescence signal under a fluorescence microscope. Therefore, SWT5015 demonstrated the optimal delivery efficiency.

[0639] Example 11: Construction of eukaryotic drug mRNA / DNA mixed dual delivery cytoplasmic vector pEU013 with GSDMD-N and presenting strains SWT5115 and SWT5119, construction of vector pEU014 with GSDME-N and presenting strains SWT5215 and SWT5219, and verification of mRNA transcription in the above strains

[0640] In this example, to verify the ability of eukaryotic drug mRNA / DNA mixed dual delivery to the cytoplasm for GSDM-N, vector pEU013 carrying GSDMD-N and presenting strains SWT5115 and SWT5119, as well as vector pEU014 carrying GSDME-N and presenting strains SWT5215 and SWT5219 were constructed.

[0641] 1. Construction of plasmid vector pEU013 and strains SWT5115 and SWT5119

[0642] The pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were used as balanced lethal control mechanisms, and a T7 promoter containing a double phage source was used to control the transcription of the drug protein GSDMD-N (SEQ ID No. 23) and T7 RNA polymerase (SEQ ID No. 20) and the expression of the membrane-breaking protein gene LLO (SEQ ID No. 13), respectively.

[0643] The specific steps are as follows:

[0644] (1) Plasmid pEU010 was digested with NcoI and XbaI to recover a fragment of approximately 7869 bp;

[0645] (2) Plasmid pEU003 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NcoI and XbaI to recover a fragment of approximately 828 bp containing the GSDMD-N sequence;

[0646] (3) The two fragments were ligated using T4 DNA ligase, transformed into competent cells of SWT1005-T7P, and plated on LB plates containing 100 μg / ml D-alanine. A single clone was selected and named SWT5115.

[0647] (4) A single clone was picked and amplified using primers SWTO129 and SWTO149; SWTO150 and SWTO151; and identified by agarose gel electrophoresis. As shown in FIG. 37 , the resulting plasmid was pEU013.

[0648] (5) Plasmid pEU013 was extracted and transformed into competent cells of SWT2009-T7P, and 100 μg / ml D-alanine LB plates were coated. A single clone was screened and named SWT5119.

[0649] 2. Construction of plasmid vector pEU014 and strains SWT5215 and SWT5219

[0650] The pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were used as balanced lethal control mechanisms, and a T7 promoter containing a double phage source was used to control the transcription of the drug protein GSDME-N (SEQ ID No. 24) and T7 RNA polymerase (SEQ ID No. 20) and the expression of the membrane-breaking protein gene LLO (SEQ ID No. 13), respectively.

[0651] The specific steps are as follows:

[0652] (1) Plasmid pEU010 was digested with NcoI and XbaI to recover a fragment of approximately 7869 bp;

[0653] (2) Plasmid pEU004 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NcoI and XbaI to recover a fragment of approximately 816 bp containing the GSDME-N sequence;

[0654] (3) The two fragments were ligated using T4 DNA ligase, transformed into SWT1005-T7P competent cells, and plated on 100 μg / ml D-alanine LB plates. A single clone was selected and named SWT5215.

[0655] (4) A single clone was picked and amplified using primers SWTO129 and SWTO152; SWTO150 and SWTO153; and identified by agarose gel electrophoresis. As shown in FIG. 37 , the resulting plasmid was pEU014.

[0656] (5) Plasmid pEU014 was extracted and transformed into competent cells of SWT2009-T7P, and 100 μg / ml D-alanine LB plates were coated. A single clone was screened and named SWT5219.

[0657] 3. Verification of mRNA transcription of GSDMD-N in strains SWT5115 and SWT5119 and GSDME-N in strains SWT5215 and SWT5219

[0658] Since SWT5115, SWT5119, SWT5215, and SWT5219 strains can all present mRNA and constitutively express T7 RNA polymerase, the target proteins eukaryotically expressed GSDMD-N and GSDME-N mRNA will be present in bacteria. Therefore, in this example, SWT1005 was used as a control, and the mRNA levels of eukaryotically expressed GSDMD-N and GSDME-N in SWT5115 and SWT5215 strains were identified by reverse transcription qPCR; at the same time, in this example, SWT2009 was used as a control, and the mRNA levels of eukaryotically expressed GSDMD-N and GSDME-N in SWT5119 and SWT5219 strains were identified by reverse transcription qPCR.

[0659] The specific steps are as follows:

[0660] (1) Single clones of SWT1005, SWT5115, SWT5215, SWT2009, SWT5119, and SWT5219 were selected and inoculated into 5 ml LB liquid culture medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0661] (2) Transfer the cultured strains into centrifuge tubes containing 1 ml of fresh corresponding culture medium at a ratio of 1:100, and culture at 32°C, 200 rpm for 4-6 hours until the OD 600 Between 0.5-1.

[0662] (3) Centrifuge the bacterial suspension of each strain at 4°C, 12,000 rpm for 2 minutes, and carefully remove all the supernatant.

[0663] (4) Resuspend the cells thoroughly (using a pipette) in 100 μl of TE buffer containing lysozyme (purchased from Shanghai Biotech Biotechnology Co., Ltd., catalog number ST206) and incubate at room temperature for 5 minutes.

[0664] (5) Add 300 μl of Lysis buffer (GeneJET RNA purification kit, purchased from Thermo, catalog number K0731) to each tube and vortex to mix.

[0665] (6) Add 180 μl of anhydrous ethanol to each tube and mix thoroughly by pipetting. Transfer all the liquid in the centrifuge tube to an RNA purification column (GeneJET RNA Purification Kit, purchased from Thermo, Cat. No. K0731), centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid. Transfer the adsorption column to a new 2 ml collection tube.

[0666] (7) Add 700 μl of wash buffer 1 (GeneJET RNA purification kit, purchased from Thermo, product number K0731) to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0667] (8) Add 600 μl of wash buffer 2 (GeneJET RNA purification kit, purchased from Thermo, product number K0731) to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0668] (9) Transfer the adsorption column to a new 1.5 ml centrifuge tube, add 50 μl of DEPC water (purchased from Shanghai Biyuntian Biotechnology Co., Ltd., product number R0022), let it stand at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, and discard the adsorption column.

[0669] (10) The RNA tube was placed on ice, and its concentration and purity were detected by Nanodrop. 500 ng was then taken and run on 1% electrophoresis to detect its integrity. The identification results are shown in FIG34A.

[0670] (11) Remove residual DNA from RNA. Pipette 1 μl of extracted RNA, add 1 μl of DNase I (purchased from Thermo, catalog number EN0521), 1 μl of DNase I buffer, and make up to 10 μl with DEPC water. Incubate at 37°C for 30 minutes and inactivate at 65°C for 10 minutes.

[0671] (12) 1 μl of RNA from the previous step was aspirated and mixed with the corresponding primers SWTO154 and SWTO155 (to identify GSDMD-N mRNA) and SWTO156 and SWTO157 (to identify GSDME-N mRNA) and Luna Universal qPCR Master Mix (purchased from NEB, catalog number M3003S) amplification enzyme system, and then the mixture was placed in the fluorescent PCR amplification device.

[0672] The amplification program was 95°C for 60 seconds; 95°C for 15 seconds, 60°C for 30 seconds, 40 cycles;

[0673] (13) No reverse transcription was performed in this step to eliminate the possibility of residual DNA interfering with the experiment. The results are shown in Figure 38.

[0674] (14) 1 μl of RNA from step (11) was aspirated and mixed with the corresponding primers SWTO154 and SWTO155 (to identify GSDMD-N mRNA) and SWTO156 and SWTO157 (to identify GSDME-N mRNA) and the amplification enzyme system of Luna Universal One-step RT-qPCR kit (purchased from NEB, product number E3005S), and then placed in the fluorescent PCR amplification device.

[0675] The amplification program was 55°C for 10 minutes; 95°C for 60 seconds; 95°C for 15 seconds, 60°C for 30 seconds, and 40 cycles. The purpose was to first convert mRNA into cDNA by reverse transcription and then amplify the target gene. The results are shown in Figure 38.

[0676] The above experimental results show that the SWT5115 and SWT5215 strains contain mRNAs transcribed from the first expression units of the target genes GSDMD-N (Figure 38A) and GSDME-N (Figure 38B), respectively, and there is no significant difference in their mRNA levels. The SWT5119 and SWT5219 strains contain mRNAs transcribed from the first expression units of the target genes GSDMD-N (Figure 38C) and GSDME-N (Figure 38D), respectively, and there is no significant difference in their mRNA levels.

[0677] The results of this example show that using different hypoxia-specific expression cassettes in the strains has no effect on the mRNA transcribed from the first expression unit.

[0678] Example 12: Verification of the pyroptotic and killing effects of strains SWT5115 and SWT5215 on various types of cancer cells

[0679] The cells were co-cultured with cancer cells under hypoxic conditions in vitro to verify the killing effects of strains SWT5115 and SWT5215 on various cancer cells, and strain SWT1005 was used as a control strain.

[0680] The specific steps are as follows:

[0681] 1. Streak strains SWT1005, SWT5115, and SWT5215 on LB plates containing 100 μg / ml D-alanine and culture them statically in a 37°C incubator overnight.

[0682] 2. Single clones of strains SWT1005, SWT5115, and SWT5215 were selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0683] 3. Measure the absorbance of the strain at 600 nm, take the culture with an OD value of 1, wash it twice with PBS, then wash it once with the corresponding cell culture medium, and then resuspend it in the cell culture medium (infection ratio is 200:1).

[0684] 4. The above bacteria were co-cultured with breast cancer (EMT6 cell line), liver cancer (Hepa1-6 cell line), lung cancer (A549 cell line), melanoma (SK-MEL-5 cell line), kidney cancer (Renca cell line), gastric cancer (MFC cell line), pancreatic cancer (Pan02 cell line), prostate cancer (RM-1 cell line), colon cancer (CT26 cell line), ovarian cancer (ID8 cell line), neuroblastoma (Neuro-2a cell line), squamous cell carcinoma (SCC7 cell line), bladder cancer (MB49 cell line), and osteosarcoma (K7M2) at an oxygen concentration below 0.8% for 2 hours. The cells were washed three times with PBS containing gentamicin, and the medium was changed and cultured in an anaerobic jar at an oxygen concentration below 0.8% for 24 hours. The cells were photographed and recorded with a 40x objective lens under white light, as shown in Figures 39-43.

[0685] 5. Mix CCK8 reagent with the above cell suspension and incubate for 1 hour.

[0686] 6. Use an enzyme-labeled instrument to measure the absorbance (detection wavelength 450 nm) and calculate the percentage of cytotoxicity.

[0687] The killing effects of strains SWT1005, SWT5115, and SWT5215 on EMT6, Hepa1-6, A549, SK-MEL-5, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7, MB49, and K7M2 cells are shown in Figure 44.

[0688] The experimental results were analyzed using T-test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0689] The above results show that the Salmonella strains SWT5115 and SWT5215 carrying the expression vector have a significant killing effect on various cancer cells. Significant cell pyroptosis can be found under a microscope under white light (indicated by arrows), while SWT1005 has no obvious pyroptosis.

[0690] In addition, analysis of the death reagent CCK8 revealed that its cell killing effect was stronger than that of the control strain SWT1005, indicating that the Salmonella GSDM-N delivered by the cytoplasm of the eukaryotic drug mRNA / DNA dual delivery has universal effects on the cell pyroptosis and killing of cancer cells.

[0691] Example 13: Verification of the pyroptotic and killing effects of strains SWT5119 and SWT5219 on various types of cancer cells

[0692] The cells were co-cultured with cancer cells under hypoxic conditions in vitro to verify the killing effects of strains SWT5119 and SWT5219 on various cancer cells, and strain SWT2009 was used as a control strain.

[0693] The specific steps are as follows:

[0694] 1. Streak strains SWT2009, SWT5119, and SWT5219 on LB plates containing 100 μg / ml D-alanine and culture them statically in a 37°C incubator overnight.

[0695] 2. Single clones of strains SWT2009, SWT5119, and SWT5219 were selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0696] 3. Measure the absorbance of the strain at 600 nm, take the culture with an OD value of 1, wash it twice with PBS, then wash it once with the corresponding cell culture medium, and then resuspend it in the cell culture medium (infection ratio is 200:1).

[0697] 4. The above bacteria were co-cultured with breast cancer (EMT6 cell line), liver cancer (Hepa1-6 cell line), lung cancer (A549 cell line), melanoma (SK-MEL-5 cell line), kidney cancer (Renca cell line), gastric cancer (MFC cell line), pancreatic cancer (Pan02 cell line), prostate cancer (RM-1 cell line), colon cancer (CT26 cell line), ovarian cancer (ID8 cell line), neuroblastoma (Neuro-2a cell line), squamous cell carcinoma (SCC7 cell line), bladder cancer (MB49 cell line), and osteosarcoma (K7M2) at an oxygen concentration below 0.8% for 2 hours. The cells were washed three times with PBS containing gentamicin, and the medium was changed and cultured in an anaerobic jar at an oxygen concentration below 0.8% for 24 hours. The cells were photographed and recorded with a 40x objective lens under white light, as shown in Figures 45-49.

[0698] 5. Mix CCK8 reagent with the above cell suspension and incubate for 1 hour.

[0699] 6. Use an enzyme-labeled instrument to measure the absorbance (detection wavelength 450 nm) and calculate the percentage of cytotoxicity.

[0700] The killing effects of strains SWT2009, SWT5119, and SWT5219 on EMT6, Hepa1-6, A549, SK-MEL-5, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7, MB49, and K7M2 cells are shown in Figure 50.

[0701] The experimental results were analyzed using T-test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0702] The above results show that the Salmonella strains SWT5119 and SWT5219 carrying the expression vector have a significant killing effect on various cancer cells. Significant cell pyroptosis can be found under a microscope under white light (indicated by arrows), while SWT2009 has no obvious pyroptosis.

[0703] In addition, analysis of the death reagent CCK8 revealed that its cell killing effect was stronger than that of the control strain SWT2009, indicating that the Salmonella GSDM-N delivered by the cytoplasm of the eukaryotic drug mRNA / DNA dual delivery has universal effects on the cell pyroptosis and killing of cancer cells.

[0704] This example also confirms that there is no difference in the killing effect on bacteria among strains using different hypoxia-specific expression cassettes, and the killing effect comes from the eukaryotic expression vector carried.

[0705] Example 14: Evaluation of the tumor suppressive properties of strains SWT5115, SWT5215 and the control strain SWT1005 in a mouse tumor model

[0706] To verify the inhibitory effect of strains carrying expression vectors on mouse tumor growth, the present invention designed SWT5115 and SWT5215 as representatives for inhibitory experiments in mouse EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7 and MB49 tumor models.

[0707] 1. Establishment of various mouse tumor models

[0708] (1) Establishment of EMT6 tumor mouse model

[0709] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6EMT6 cancer cells were used to establish a mouse breast subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0710] (1) Establishment of K7M2 tumor mouse model

[0711] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 K7M2 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0712] (2) Establishment of Hepa1-6 tumor mouse model

[0713] C57BL / 6 mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Hepa1-6 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0714] (3) Establishment of A549 tumor mouse model

[0715] Nu / Nu nude mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 A549 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0716] (4) Establishment of B16F10 tumor mouse model

[0717] C57BL / 6 mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 B16F10 cancer cells were used to establish a mouse breast subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0718] (5) Establishment of Renca tumor mouse model

[0719] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Renca cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0720] (6) Establishment of MFC tumor mouse model

[0721] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 MFC cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0722] (7) Establishment of Pan02 tumor mouse model

[0723] C57BL / 6 mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Pan02 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0724] (8) Establishment of RM-1 tumor mouse model

[0725] C57BL / 6 mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 RM-1 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0726] (9) Establishment of CT26 tumor mouse model

[0727] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 CT26 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0728] (10) Establishment of ID8 tumor mouse model

[0729] C57BL / 6 mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 ID8 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0730] (11) Establishment of Neuro-2a tumor mouse model

[0731] A / J mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Neuro-2a cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0732] (12) Establishment of SCC7 tumor mouse model

[0733] Nu / Nu nude mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 SCC7 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0734] (13) Establishment of MB49 tumor mouse model

[0735] C57BL / 6 mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 MB49 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT1005, SWT5115, and SWT5215 groups, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0736] 2. Bacterial preparation and treatment

[0737] The constructed Salmonella strains SWT1005, SWT5115, and SWT5215 were selected and inoculated into 5 ml of LB liquid medium supplemented with D-alanine and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The absorbance of the strains at 600 nm was measured, and the culture with an OD value of 1 was measured and washed three times with PBS. 1 × 10 7 CFU count bacteria were administered to model mice; the day of administration was designated as day 0, and the length and width of the mouse tumors were measured on days 1, 3, 5, 7, 9, and 11 of administration, and the mouse tumor volume was calculated according to the following formula: Mouse tumor volume = length × width 2 ×0.52

[0738] The tumor inhibitory effects of SWT1005, SWT5115, and SWT5215 in mouse tumor models are shown in Figures 51-54.

[0739] The experimental results were analyzed using T-test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0740] The results showed that the Salmonella strains SWT5115 and SWT5215 carrying the expression vector had a significant inhibitory effect on tumor growth, which was better than the control strain SWT1005 and the untreated control.

[0741] The information of the corresponding strains of the present invention is shown in Table 3

[0742] Table 3 Strain construction table

[0743] The tool plasmids of the present invention are shown in Table 4.

[0744] Table 4 Tool plasmids used in the present invention

[0745] The sequences of promoter, gene coding region, protein, etc. used in the present invention are shown in Table 5.

[0746] Table 5 Specific sequences of promoters, gene coding regions, proteins, etc. used in the present invention

[0747] The specific sequences of all primers used in the present invention are shown in Table 6.

[0748] Table 6 Specific sequences of all primers used in the present invention

[0749] The present application has been described above in conjunction with preferred embodiments, but these embodiments are merely exemplary and serve only as an illustrative example. On this basis, various replacements and improvements can be made to the present application, all of which fall within the scope of protection of the present application.

Claims

1. A eukaryotic expression vector comprising a first expression unit and a second expression unit, wherein the first expression unit comprises a first promoter derived from a bacteriophage, a eukaryotic drug protein gene expression module, and a bacteriophage-derived RNA polymerase gene transcription module, and the second expression unit comprises a second promoter derived from a bacteriophage and a membrane-breaking protein gene expression module, the first promoter controls the expression of the eukaryotic drug protein gene and the transcription of the bacteriophage-derived RNA polymerase gene, respectively, and the second promoter controls the expression of the membrane-breaking protein gene, and the bacteriophage-derived promoter binds to the RNA polymerase encoded by the specific phage RNA polymerase gene on the host bacterial chromosome.

2. The eukaryotic expression vector according to claim 1, wherein the first promoter and the second promoter derived from phage are selected from T7, T3 and SP6 promoters. The eukaryotic expression vector according to claim 2 , wherein the first promoter and the second promoter derived from bacteriophage are T7 promoters.

4. The eukaryotic expression vector according to any one of claims 1 to 3, wherein the eukaryotic drug protein gene expression module comprises a sequentially expressed phage-derived promoter IRES sequence, a Kozak sequence, the ORF region and the 3'-UTR region of the eukaryotic drug protein gene, and its product mRNA comprises the IRES sequence, the Kozak sequence, the ORF region and the 3'-UTR region of the eukaryotic drug protein gene.

5. The eukaryotic expression vector according to any one of the preceding claims, wherein the phage-derived RNA polymerase gene transcription module comprises a sequentially expressed phage-derived promoter IRES sequence, a kozak sequence, an orf region of a specific phage RNA polymerase gene, a polyA tail, and a T7 terminator sequence, and its product mRNA comprises a sequentially expressed IRES sequence, a kozak sequence, an orf region of a specific phage RNA polymerase gene, and a polyA tail.

6. The eukaryotic expression vector according to any one of the preceding claims, comprising the essential gene asd of Salmonella, and wherein the asd gene on the chromosome of the host bacterium is deleted.

7. The eukaryotic expression vector according to any one of the preceding claims, comprising a replicon essential for plasmid replication. The eukaryotic expression vector according to claim 7 , wherein the replicon is selected from the group consisting of pUC, p15A, ColE1 and R6K. The eukaryotic expression vector according to claim 8 , wherein the replicon is pUC.

10. The eukaryotic expression vector according to any one of the preceding claims, wherein the drug protein gene is an N-terminal fragment of a drug gene that causes cell pyroptosis and is derived from eukaryotic cells.

11. The eukaryotic expression vector according to claim 10, wherein the drug protein gene is selected from the N-terminus of GSDM-A, the N-terminus of GSDM-B, the N-terminus of GSDM-C, the N-terminus of GSDM-D and the N-terminus of GSDM-E.

12. The eukaryotic expression vector according to claim 11, wherein the drug protein gene is the N-terminus of GSDM-D.

13. The eukaryotic expression vector according to claim 11, wherein the drug protein gene is the N-terminus of GSDM-E.

14. The eukaryotic expression vector according to any one of the preceding claims, wherein the membrane permeabilization protein gene is selected from the listeriolysin hlyA gene.

15. The eukaryotic expression vector according to any of the preceding claims, wherein the eukaryotic expression vector is presented in a mixed mRNA / DNA cytoplasmic expression mode, i.e., the eukaryotic expression vector DNA and the mRNA produced by transcription of the first expression unit are mixed and presented into eukaryotic cells by host bacteria, and cytoplasmic expression is achieved.

16. The eukaryotic expression vector according to claim 15, which adopts a "self-reinforcement" mode, that is, the mRNA transcribed from the first expression unit is translated into T7 RNA polymerase in the eukaryotic cytoplasm, which acts on the eukaryotic expression vector DNA to achieve transcription and translation of the target gene in the cytoplasm, and further enhances the expression of T7 RNA polymerase, forming a cycle.

17. The eukaryotic expression vector according to any one of the preceding claims, wherein the host bacterium is a Gram-negative bacterium.

18. A method for constructing the eukaryotic expression vector according to any one of the above claims, comprising the following steps: (1) sequentially connecting the phage-derived first promoter, the eukaryotic drug protein gene expression module, and the phage-derived RNA polymerase gene transcription module according to any of the above claims to construct a first expression unit; (2) sequentially connecting the second phage-derived promoter and the membrane-permeabilizing protein gene expression module according to any of the above claims to construct a second expression unit; (3) The plasmid replicon, asd gene, and the above two expression units are connected together to form a complete eukaryotic expression vector.

19. A modified Gram-negative bacterium comprising the eukaryotic expression vector according to any one of the above claims. 20 . The Gram-negative bacterium according to claim 19 , wherein the specific phage RNA polymerase gene is a gene corresponding to a first promoter and a second promoter derived from a phage.

21. The Gram-negative bacterium according to claim 20, wherein the specific bacteriophage RNA polymerase gene is selected from the group consisting of a T7 RNA polymerase gene, a T3 RNA polymerase gene, and an SP6 RNA polymerase gene. The Gram-negative bacterium according to claim 21 , wherein the specific bacteriophage RNA polymerase gene is a T7 RNA polymerase gene.

23. The Gram-negative bacterium according to any one of claims 19 to 22, wherein the expression of the specific bacteriophage RNA polymerase gene is regulated by a constitutive expression promoter. The Gram-negative bacterium according to claim 23 , wherein the constitutive expression promoter is a lacUV5 promoter.

25. The Gram-negative bacterium according to any one of claims 19 to 24, further comprising a hypoxia-specific gene expression cassette, wherein the hypoxia-specific gene expression cassette comprises: a) a positive hypoxia promoter, which is a promoter comprising an FNR binding site, and the positive hypoxia promoter can be induced to express under hypoxia; b) genes essential for survival; and c) an inverted hyperoxic promoter, which is a promoter comprising FNR and ArcA binding sites, and the inverted hyperoxic promoter can function under the oxygen content conditions of normal organs; The essential gene for survival is a gene encoding alanine racemase.

26. The Gram-negative bacterium according to claim 25, wherein: The FNR binding site of the forward hypoxia promoter or the reverse hyperxia promoter conforms to the pattern of TTGATNNNNATCAA, where N is any base among A, T, C, and G. Any base in the TTGAT and ATCAA sequences in the conserved binding site can be replaced, but the total number of replacements does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; The ArcA binding site of the inverted hyperoxia promoter conforms to the GTTAATTA pattern, in which any base can be replaced, but the total number of replacements does not exceed 2. 27 . The Gram-negative bacterium according to claim 25 , wherein the forward hypoxia promoter and / or the reverse hyperoxia promoter of the hypoxia-specific gene expression cassette are promoters derived from Gram-negative bacteria.

28. The Gram-negative bacterium according to any one of claims 25 to 27, wherein The positive hypoxia promoter is selected from the promoter region sequence of yhbU or ynfK; Genes essential for survival are selected from alr or dadX; The inverted hyperoxia promoter is selected from the promoter region sequence of cyoA or ydcI.

29. Gram-negative bacteria according to claim 28, wherein the positive hypoxia promoter is yhbU, which is selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia and Simihuiya.

30. The Gram-negative bacterium according to claim 28, wherein the positive hypoxia promoter is ynfK, which is selected from Salmonella, Escherichia coli, Serratia, Shigella and Enterobacter reuteri.

31. The Gram-negative bacterium according to claim 28, wherein the alanine racemase gene alr and / or dadX is selected from Salmonella, Escherichia coli, Shigella, Klebsiella, Yersinia, Haemophilus and Pseudomonas.

32. The Gram-negative bacterium according to claim 28, wherein the hypoxia-specific gene expression cassette consists of a forward hypoxia promoter yhbU, a survival essential gene alr, and a reverse hyperoxia promoter cyoA.

33. The Gram-negative bacterium according to claim 28, wherein the hypoxia-specific gene expression cassette consists of a forward hypoxia promoter yhbU, a survival essential gene alr, and a reverse hyperoxia promoter ydcI. The Gram-negative bacterium according to claim 28 , wherein the hypoxia-specific gene expression cassette consists of a positive hypoxia promoter ynfK, a survival essential gene dadX, and a negative hyperoxia promoter cyoA. The Gram-negative bacterium according to claim 28 , wherein the hypoxia-specific gene expression cassette consists of a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperoxia promoter ydcI.

36. The Gram-negative bacterium according to any one of claims 25 to 35, wherein the hypoxia-specific gene expression cassette is regulated by oxygen concentration.

37. The Gram-negative bacterium according to claim 36, wherein the forward hypoxic promoter functions when the oxygen content is lower than 1%, but fails to function when the oxygen content is higher than 1%; and / or the reverse hyperoxic promoter functions when the oxygen content is higher than 1%, but fails to function when the oxygen content is lower than 1%.

38. The Gram-negative bacterium according to claim 37, wherein the forward hypoxic promoter functions when the oxygen content is lower than 0.8%, but fails to function when the oxygen content is higher than 0.8%; and / or the reverse hyperoxic promoter functions when the oxygen content is higher than 0.8%, but fails to function when the oxygen content is lower than 0.8%.

39. A method for controlling the expression of a pharmaceutical protein in prokaryotic cells using the eukaryotic expression vector according to any one of claims 1 to 17, comprising the following steps: (1) preparing the eukaryotic expression vector according to any one of the above claims; (2) integrating an expression cassette constitutively expressing a bacteriophage RNA polymerase gene into the host bacterial chromosome; (3) knocking out the asd gene on the host bacterial chromosome; (4) Transforming the eukaryotic expression vector according to any one of the above claims into a host bacterium.

40. The method of claim 39, further comprising integrating the hypoxia-specific gene expression cassette of any one of claims 25-38 into a host bacterial chromosome.

41. The method of claim 39 or 40, wherein the host bacterium is a Gram-negative bacterium.

42. The Gram-negative bacteria according to any one of the preceding claims, which is selected from the group consisting of Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simihuiella, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, Vibrio parahaemolyticus, Vibrio cholerae, Yersinia pestis, Coccus catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella spp., Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori. The Gram-negative bacterium according to claim 42 , which is Salmonella.

44. The Gram-negative bacterium according to claim 42 or 43, which is an attenuated Gram-negative bacterium.

45. The method of claim 44, wherein the attenuated Gram-negative bacterium is Salmonella.

46. The Gram-negative bacterium according to any of the preceding claims, wherein the mode of presenting the eukaryotic expression vector is a mixed mRNA / DNA cytoplasmic expression mode, i.e., the eukaryotic expression vector DNA and the mRNA produced by transcription of the first expression unit are mixed and presented into eukaryotic cells by the host bacteria, and cytoplasmic expression is achieved.

47. A pharmaceutical composition comprising the eukaryotic expression vector according to any one of the preceding claims, or the Gram-negative bacteria according to any one of the preceding claims.

48. The pharmaceutical composition according to claim 47, further comprising a pharmaceutically acceptable carrier.

49. The pharmaceutical composition of claim 48, wherein the pharmaceutically acceptable carrier is selected from the group consisting of disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants, and lubricants.

50. The pharmaceutical composition according to any one of claims 47 to 49, for use in the treatment of solid tumors.

51. Use of the eukaryotic expression vector according to any one of the above claims, the Gram-negative bacteria according to any one of the above claims, or the pharmaceutical composition according to any one of the above claims in the preparation of an anti-tumor drug.

52. The use according to claim 51, wherein the tumor is a solid tumor.

53. The pharmaceutical composition of claim 50 or the use of claim 52, wherein the solid tumor is selected from the group consisting of breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal (colon cancer, rectal cancer), pelvic (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer, and bladder tumors / cancers.

54. The pharmaceutical composition or use according to claim 53, wherein the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

55. A method for treating tumors, comprising administering to a subject the eukaryotic expression vector according to any one of the above claims, the Gram-negative bacteria according to any one of the above claims, or the pharmaceutical composition according to any one of the above claims.

56. The method of claim 55, wherein the tumor is a solid tumor.

57. The method of claim 56, wherein the solid tumor is selected from the group consisting of tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal (colon cancer, rectal cancer), pelvic (cervical cancer, ovarian malignancies, endometrial cancer, ovarian cancer), nervous system, head and neck cancer, and bladder.

58. The method of claim 57, wherein the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma, and bladder cancer.

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