Eukaryotic expression vector, bacteria containing eukaryotic expression vector and application of eukaryotic expression vector

Abstract

The invention belongs to the field of genetic engineering and biological treatment, and provides an eukaryotic expression vector, gram-negative bacteria containing the eukaryotic expression vector and application of the eukaryotic expression vector in preparation of tumor drugs. By selecting and combining a phage-derived promoter, a specific phage RNA polymerase gene, a membrane rupture protein gene and an eukaryotic expression drug protein gene, an eukaryotic expression vector is divided into three modules: (1) a phage-derived RNA polymerase gene transcription module; (2) a membrane rupture protein gene expression module; and (3) a eukaryotic drug protein gene expression module. Through organic combination of the three modules, the eukaryotic drug delivery system is constructed, and the system and method for stably and efficiently performing eukaryotic drug protein expression, drug delivery and drug killing are realized.

Description

Technical Field

[0001] The present invention relates to a eukaryotic expression vector, a bacterium containing the same, and applications thereof, and particularly to a constitutive eukaryotic drug mRNA / DNA hybrid dual-delivery cytoplasmic expression system containing a dual-phage-derived promoter, a bacterium containing the same, and applications thereof, belonging to the fields of genetic engineering and biotherapy. Background Art

[0002] Cancer is one of the main causes of death worldwide, and the prevalence of cancer is increasing annually. Among all malignant tumors, solid tumors account for approximately 90%, such as sarcoma, melanoma, breast cancer, lung cancer, colon cancer, prostate cancer, etc.

[0003] The tumor microenvironment of solid tumors has common characteristics, including abnormal tumor vascular systems, excessive connective tissues, immunosuppression, acidic environments, and hypoxic regions, etc. In addition, the abnormal microenvironment of solid tumors is a natural barrier that is difficult for traditional therapeutic drugs to penetrate, and chemotherapy drugs and antibody drugs are difficult to diffuse within the solid tumor microenvironment. Moreover, the lack of oxygen free radicals makes tumors resistant to chemotherapy and radiotherapy.

[0004] Due to the hypoxic environment within solid tumors, facultative anaerobes and obligate anaerobes can invade tumors and inhibit their growth, and thus can be used as therapeutic agents or carriers with great potential.

[0005] As early as 1868, German doctor W. Busch first reported that in some cancer patients, while being infected with bacteria (Streptococcus pneumoniae), the growth of tumors in their bodies was inhibited, and even completely cleared. In the following thirty years, American doctor Coley and another German doctor Fehleisen separately reported the phenomenon that bacterial infection could inhibit tumors. These early research works have always been controversial because their results were difficult to replicate, and the virulence of bacteria was also difficult to control. However, later rigorous animal experiments proved that the invasion of bacteria could indeed reduce the size of tumors, and the host immune system would also be activated during the treatment process. In 1975, Carswell first reported that the endotoxin (lipopolysaccharide) of Gram-negative bacteria could stimulate the immune system to release tumor necrosis factor TNF-α, and could cause the death of tumor cells. In addition, some bacterial vaccines have also been proven to stimulate the immune system to treat tumors. Among them, Bacillus Calmette-Guérin (BCG) is the earliest biological agent applied to clinical tumor treatment. BCG is a live attenuated bacterial suspension prepared by continuously passaging the highly pathogenic Mycobacterium bovis for 230 generations, and its purpose is for the prevention of tuberculosis. Through a large number of experiments and clinical practices, it has been confirmed that BCG is one of the most effective means for treating bladder cancer.

[0006] In recent years, with the rapid development of molecular biology and genetic engineering technologies, it has been found that some facultative or obligate anaerobic bacteria can target, colonize and proliferate in solid tumors and induce tumor regression. For example, obligate anaerobic bacteria such as Clostridium, and probiotic Bifidobacterium. Among them, the Gram-negative facultative anaerobic bacterium Salmonella enterica has the most extensive application prospects.

[0007] Studies have shown that after attenuating Salmonella typhimurium by different methods, its colonization ability in tumor tissues can reach 1000-10000 times that of normal tissues, and it can be used as a potential targeted drug for treating tumors.

[0008] Regarding the improvement of recombinant bacteria targeting tumor regions, there have been reports in the literature that these recombinant bacteria can also carry different types of drugs to achieve the effect of targeted drug delivery to tumor regions. However, the effect is not ideal. The reason may be that the drug synthesis and delivery efficiency of bacteria in tumors have not been comprehensively considered.

[0009] In addition, since bacteria are used as carriers for drugs, the drugs carried are restricted by the conditions of the bacteria themselves. First of all, bacteria are prokaryotes, and the proteins directly produced by their expression systems lack glycosylation modification and systems such as chaperone proteins necessary for protein folding. Therefore, if the drugs carried belong to eukaryotic cell proteins, it is difficult to express correctly folded or glycosylated proteins, thus affecting their efficacy. Secondly, if the expressed drug protein is toxic to bacteria, the bacteria cannot express it efficiently.

[0010] Therefore, when using bacteria as carriers to carry eukaryotic-expressed drug genes and deliver them to eukaryotic cells for expression, since the DNA transcription program of the eukaryotic system occurs in the nucleus, if DNA is directly delivered by bacteria, these DNAs need to shuttle into the nucleus. However, the probability of foreign DNA entering the nucleus is very low. Only during cell mitosis, the nuclear membrane of the nucleus will depolymerize, and foreign DNA may enter the nuclear region. Therefore, the cytoplasmic expression mode is the key to increasing the delivery efficiency.

[0011] The 2023 Nobel Prize in Physiology or Medicine was awarded to scientists Katalin Karikó and Drew Weissman in recognition of their contributions to nucleobase modification. Their contributions have made the development of effective messenger ribonucleic acid (mRNA) vaccines against COVID-19 possible. The success of mRNA vaccines compared to DNA vaccines suggests that the direct cytoplasmic expression mode of mRNA has significant advantages compared to the DNA expression mode that needs to enter the nucleus.

[0012] U.S. Patent US10987432B2 reports a method for cytoplasmic expression based on a bacterial vector. The method is to deliver a DNA / mRNA hybrid vector by Salmonella. The vector carries a drug gene controlled by a CMV promoter and a T7 promoter, and at the same time, an autogene composed of a T7 promoter and a T7 RNA polymerase gene is carried downstream to automatically enhance gene expression. The purpose is to continuously synthesize T7 RNA polymerase in the cytoplasm through the self-carried T7 RNA polymerase gene, forming a positive feedback gene circuit, and at the same time, the target drug gene can be synthesized.

[0013] However, this design is not a truly cytoplasmic expression vector in essence. In this design, it carries a CMV promoter (a transcriptional promoter in the nucleus) and at the same time carries a signal regulatory element of polyA (this element needs to participate in the mRNA maturation process in the nucleus) instead of polyadenine, and it cannot effectively form a polyA mRNA tail in the cytoplasm. Therefore, it is not a specific and effective cytoplasmic expression method, but belongs to a cytoplasmic expression mode that depends on nuclear entry.

[0014] In addition, on the drug gene vector, two T7 promoters regulate the transcription of the drug gene and T7 RNA polymerase respectively. This design may cause mRNA diversity after transcription, ultimately resulting in a reduction in the number of effective mRNAs delivered to the cytoplasm. At the same time, the expression pattern of its membrane-breaking protein has not been optimized, which will affect its final delivery efficiency and thus affect the overall therapeutic effect.

[0015] In summary, there is an urgent need for a eukaryotic drug protein system and method that can stably and efficiently express and release in in-vivo tumors of bacteria. Summary of the Invention

[0016] In the present invention, unless otherwise specified, the scientific and technical terms used in the present invention have the meanings commonly understood by those skilled in the art. And the relevant terms and laboratory operation steps of protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in the present invention are all widely used terms and conventional steps in the corresponding fields.

[0017] In view of the above-mentioned defects existing in the prior art, the present invention has established a constitutive eukaryotic drug mRNA / DNA hybrid dual-delivery cytoplasmic expression system containing a dual-phage-derived promoter.

[0018] To this end, the present invention provides a constitutive eukaryotic drug mRNA / DNA hybrid dual-delivery cytoplasmic expression system containing a dual-phage-derived promoter, which comprises three modules: (1) a phage-derived promoter and RNA polymerase transcription control module; (2) a delivery module; (3) a eukaryotic drug mRNA transcription module and a phage-derived RNA polymerase self-enhancing module. By the organic combination of the three modules and in line with the characteristics of bacteria, the problem that bacterial vectors are difficult to carry eukaryotic drugs is solved.

[0019] 1. Phage-derived promoter and RNA polymerase transcription control module

[0020] In order to achieve stable and efficient gene expression in the tumor microenvironment, a high-intensity promoter expression system that is not affected by the environment needs to be used. The present invention uses 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 bacteria to achieve stable inheritance. Since T7 RNA polymerase specifically recognizes the T7 promoter, stable and high-intensity expression of genes controlled by the T7 promoter can be achieved. Therefore, the delivery module, the eukaryotic drug mRNA transcription module, and the phage-derived RNA polymerase self-enhancing module are respectively placed behind the T7 promoter and expressed and regulated on a plasmid.

[0021] 2. Delivery module

[0022] When bacteria invade tumor cells, phagocytic vesicles will be formed. The existence of phagocytic vesicles will limit the further release of bacteria and the drugs they synthesize inside the cells. To solve this problem, the present invention uses listeriolysin O (LLO) as a membrane-breaking protein to achieve the purpose of drug delivery. To increase its efficiency as a membrane-breaking protein, the present invention places the LLO gene on an expression vector plasmid and uses the T7 promoter to control its expression. This not only increases the overall expression level of LLO, but also, by expressing it synchronously with the mRNA of eukaryotic drugs in advance, can efficiently break the membrane while phagocytic vesicles are being formed, and improves the overall presentation efficiency compared with Patent US10987432B2.

[0023] 3. Eukaryotic drug mRNA transcription module and phage-derived RNA polymerase self-enhancing module

[0024] (1) Selection of eukaryotic drugs

[0025] To achieve a precision drug that can effectively kill tumor cells only, the present invention uses the N-terminal fragment of the eukaryotic pyroptosis protein. Pyroptosis is a form of cell death that is crucial for immunity. It is typically induced by the canonical caspase-1 inflammasome or activated caspase-4, -5, and -11. The activated caspase cleaves the pyroptosis protein gasdermin (including GSDMA, GSDMB, GSDMC, GSDMD, GSDME) in the linker region of the gasdermin protein to release its gasdermin-N-terminal fragment domain, which is in an activated state. This fragment can penetrate the cell membrane, thereby inducing pyroptosis. Studies have found that after caspase activation, GSDMD is proteolytically cleaved at the Asp 275 site, while GSDME is specifically cleaved by caspase-3 at the Asp 270 site into two parts and becomes active. The N-terminal protein of the cleaved GSDMD or GSDME protein has pore-forming activity and can insert into the cell membrane to form pores, thereby triggering pyroptosis. GSDME is silenced in most cancer cells but is expressed in many normal tissues. After chemotherapy drugs activate caspase-3, human primary cells exhibit GSDME-dependent pyroptosis. The C-terminal of GSDMD or GSDME acts as an inhibitor and inhibits the activity of the N-terminal in the non-cleaved state. The N-terminal cleavage product of GSDM localizes to the plasma membrane by anchoring to membrane lipids and specifically interacts with phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] in the inner leaflet of the mammalian cell membrane. 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 intramembrane polymerization, forming wide pores with an inner diameter of 10-14 nm. The osmotic pressure is disrupted due to pore formation, leading to cell swelling and lysis, which are the morphological characteristics of pyroptosis. These wide pores also act as protein secretion channels, promoting the secretion of inflammatory cytokines, thus achieving a rapid innate immune response. Since [PI(4)P] and [PI(4,5)P] are absent outside the cell, GSDM-N leaked outside the cell is non-toxic. Since the action of GSDM-N on the inner cell membrane is non-discriminatory, GSDM-N is also toxic to bacteria.

[0026] Two characteristics of GSDM-N: It is derived from eukaryotic cells, requires glycosylation modification, and its non-discriminatory attack on the bacterial inner membrane makes it impossible to directly express the GSDM-N gene in bacteria. Therefore, instead of directly expressing it in bacteria, using a eukaryotic system for presentation is a very good strategy.

[0027] (2) Selection of the eukaryotic mRNA cytoplasmic expression system

[0028] Studies have shown that Salmonella can carry eukaryotic plasmids and can deliver eukaryotic plasmids into cells. However, since plasmid DNA needs to enter the nucleus to perform transcription into mRNA and then start the translation process, the efficiency is very low. To solve this problem, the present invention uses a bacterial system to directly synthesize eukaryotic mRNA in bacteria, and through a delivery system, the mRNA / DNA vector can be directly delivered into the cytoplasm of cancer cells, and eukaryotic drug proteins are synthesized by using the translation system of the cytoplasm. The structure of mature mRNA includes the viral internal ribosome entry site (IRES), kozak sequence, orf region of the eukaryotic drug, 3'-UTR region, and polyA tail. In the present invention, since bacteria themselves lack the enzymes related to mRNA capping, the viral internal ribosome entry site IRES is used instead, and bacteria can successfully synthesize and carry mRNA containing IRES and deliver it to the cytoplasm of cancer cells.

[0029] (3) Phage-derived RNA polymerase self-amplifying system based on mRNA / DNA hybrid expression

[0030] Although bacteria can directly deliver the synthesized mRNA into the cytoplasm for expression, due to the low efficiency of bacteria in delivering nucleic acids, it needs to be further improved. In addition to adjusting the spatio-temporal expression pattern of LLO as described above, the method of the present invention is further enhanced by phage-derived RNA polymerase self-amplification. Compared with the method of patent US10987432B2, this patent completely abandons the nuclear expression and the mode of two monocistrons (self-amplifying transcription controlled by a dual T7 promoter), and adopts a method of pure cytoplasmic expression. 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 mRNA maturation process in the nucleus) is discarded. Instead, it is directly designed into a mode of single mRNA bicistronic transcription. After a single T7 promoter, IRES is directly connected to control the stable expression of the drug gene and 3'-UTR, and then a second IRES controls the stable expression of T7 RNA polymerase and 3'-UTR, and a polyA sequence is directly used as the 3' end of mRNA downstream. This design can achieve an efficient mode of mRNA / DNA hybrid delivery.

[0031] During the tumor treatment process of the present invention, bacteria enter tumor tissue through the circulatory system and colonize there. Stable expression of the T7 RNA polymerase within the bacteria promotes the massive synthesis of the GSDM-N fragment and bicistronic mRNA for the 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, 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, the bicistronic mRNA for the T7 RNA polymerase, and the 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 elimination 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.

[0032] 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.

[0033] 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).

[0034] In a more preferred embodiment of the present invention, the dual phage-derived promoter is the T7 promoter (SEQ ID No. 1).

[0035] 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.

[0036] 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 the T7 RNA polymerase protein sequence (SEQ ID No.5) optimized by Salmonella codons, the T3 RNA polymerase gene (SEQ ID No.6) and the T3 RNA polymerase protein sequence (SEQ ID No.7), and the SP6 RNA polymerase gene (SEQ ID No.8) and the SP6 RNA polymerase protein sequence (SEQ ID No.9).

[0037] In a further preferred embodiment of the present invention, the specific phage RNA polymerase gene is the T7 RNA polymerase gene.

[0038] In order to express the pharmaceutical protein stably and efficiently, in the present invention, the pharmaceutical protein gene is placed on a multi-copy expression plasmid vector and controlled by the T7 promoter to achieve the maximum intensity of expressing the pharmaceutical protein gene.

[0039] In order to achieve a higher expression intensity, in a preferred embodiment of the present invention, an RNA polymerase expression system of T7 phage is adopted, which includes T7 RNA polymerase and a T7 promoter to control the expression of the target pharmaceutical protein.

[0040] The T7 RNA polymerase system is derived from the expression system of Escherichia coli T7 phage and is widely used in the gene expression of Escherichia coli due to its strong protein expression ability. The characteristic of T7 RNA polymerase is that it can specifically recognize the T7 promoter and is not affected by environmental factors. At the same time, the RNA synthesis rate of T7 RNA polymerase is five times that of Escherichia coli RNA polymerase, so it can highly express the target protein. In addition, the transcription of T7 RNA polymerase can be independently executed without the assistance of other transcription factors, so it can independently initiate transcription even inside the cytoplasm of eukaryotic cells. It has great advantages for the cytoplasmic expression system.

[0041] In order to achieve stable expression without being affected by the internal metabolism of bacteria and the microenvironment in tumors, in a more preferred embodiment of the present invention, the constitutive expression lacUV5 promoter (SEQ ID No.10) is further adopted to control the expression of the specific phage RNA polymerase gene.

[0042] The lacUV5 promoter is very similar to the classical lac promoter. Compared with the lac promoter, it contains only 2 base pair mutations in the -10 region. The LacUV5 promoter does not require an additional activator and can drive high levels of gene expression. Although no activator is required, the expression of the lacUV5 promoter in Escherichia coli can be regulated by the LacI repressor and induced by IPTG. IPTG is an effective inducer when used at concentrations in the range of 100 μM to 1.5 mM.

[0043] Since Salmonella lost the lacI gene and the entire lac operon during evolution, the use of the lacUV5 promoter in Salmonella is an excellent constitutive expression system.

[0044] Therefore, in a more preferred embodiment of the present invention, the T7 RNA polymerase expression cassette controlled by the lacUV5 promoter was further transferred into the chromosome of Salmonella, thus achieving stable constitutive expression.

[0045] In a preferred embodiment of the present invention, the T7 polymerase and the T7 promoter can alternatively be replaced with the T3 polymerase and the T3 promoter. Similar to the T7 RNA polymerase system, the T3 RNA polymerase system is an RNA polymerase system derived from phage T3 that highly specifically recognizes the T3 promoter sequence. Or the T7 polymerase and the T7 promoter can alternatively be replaced with the SP6 polymerase and the SP6 promoter. Similar to the T7 RNA polymerase system, the SP6 polymerase is also an RNA polymerase system derived from phage SP6 that highly specifically recognizes the SP6 promoter sequence. The T3 RNA polymerase or the SP6 RNA polymerase and their corresponding promoters can functionally replace the T7 RNA polymerase to specifically control the expression of downstream target genes.

[0046] In a preferred embodiment of the present invention, a drug protein and T7 RNA polymerase are expressed in a eukaryotic manner in tandem with a T7 promoter bicistron. There are two advantages: First, inside the bacteria, the T7 polymerase transcribes the drug protein and T7 RNA polymerase to produce a single bicistronic mRNA product. The single product is more conducive to controlling the ratio of the drug protein and T7 RNA polymerase to achieve the best presentation efficiency. Second, directly controlling the T7 RNA polymerase with the T7 promoter is likely to generate a continuous positive feedback self-reinforcing cycle, resulting in the depletion of bacterial internal resources and causing bacterial death. Although the T7 RNA polymerase used on the vector is in a eukaryotic expression mode and lacks the essential RBS, which can reduce the efficiency of prokaryotic expression in bacteria, it cannot completely avoid leaky expression. Therefore, the self-reinforcing design of the bicistron will increase the overall bacterial viability and efficiency.

[0047] In addition, to prevent the loss of the plasmid expression vector, a necessary gene of Salmonella needs to be implanted during the construction of the vector, and at the same time, this gene is removed from the chromosome of the host bacterium, thus forming a balanced lethal control mechanism. That is, if Salmonella loses the carried plasmid vector, the bacterium will quickly die due to the lack of the necessary gene, and the surviving bacteria all carry the plasmid expression vector, thereby ensuring the stable and efficient expression of protein drugs in the whole system. The asd gene (SEQ ID No. 11) of Salmonella and the protein sequence of asd (SEQ ID No. 12) encode aspartate B-semialdehyde dehydrogenase, which is an enzyme required in the process of synthesizing diaminopimelic acid (DAP), an important component of the cell wall of Gram-negative bacteria. Knocking out the asd gene of Salmonella will lead to the lysis and death of Salmonella. However, when DAP is additionally supplemented in the culture medium or Salmonella is made to carry a vector containing the asd gene, the normal growth of Salmonella can be maintained. Therefore, asd is used as a necessary gene for the plasmid vector in the present invention. In a preferred embodiment of the present invention, the expression vector contains the necessary gene asd of Salmonella.

[0048] In order to perform drug delivery stably and efficiently, the present invention designs a precise drug delivery system. Its goal is to precisely release the drug protein synthesized by bacteria into tumor cells, which is further divided into two steps: bacteria invading host cancer cells and releasing the synthesized drug protein.

[0049] In a preferred embodiment of the present invention, the natural ability of Salmonella to invade host animal cells is utilized to achieve the invasion of host cancer cells by bacteria.

[0050] Salmonella enterica can invade host cells and replicate within host cells, including host epithelial cells and macrophages. This ability exists in pathogenicity island 1 of Salmonella. Through type III secretion system 1 (T3SS1), Salmonella secretes T3SS effector proteins, inducing a large amount of actin rearrangement in host cells, resulting in membrane ruffling, and thus forming phagocytic vesicles containing Salmonella. Then, the survival and proliferation of Salmonella in phagocytic vesicles are promoted by type III secretion system 2 (T3SS2) encoded by Salmonella pathogenicity island 2 (SPI-2) genes.

[0051] In a preferred embodiment of the present invention, the purpose of releasing the synthesized drug protein is achieved by using the listeriolysin-O (LLO) gene (SEQ ID No. 13) and protein sequence (SEQ ID No. 14) of Salmonella, which are synthesized by Salmonella and optimized by Salmonella codons.

[0052] LLO is encoded by the hlyA gene of Listeria monocytogenes (LM), and can bind to cholesterol on the host cell membrane to form a pore-like structure with a diameter of 35 nm.

[0053] LLO has an acidic domain sensitive to pH. The optimal pH for the maturation of the LLO precursor protein and the onset of its membrane-perforating activity is approximately 5.0 - 5.5, and it is inactivated at neutral pH.

[0054] When Salmonella invades and forms a phagosome, the phagosome will fuse with intracellular vacuoles such as lysosomes, thereby greatly reducing the pH. Under this condition, the activity of LLO is activated and it plays a perforating role, enabling Salmonella to release the synthesized drug protein.

[0055] Therefore, in a preferred embodiment of the present invention, the membrane-breaking protein gene is the Listeriolysin hlyA gene.

[0056] 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 and is induced to start expressing after the bacteria invade the cell and form a phagosome. However, its expression level and efficiency have a serious lag effect compared to the vector that needs to be delivered. Therefore, in the present invention, the same T7 promoter is used to control the expression of LLO. This can ensure the synchronous transcription and release of LLO and eukaryotic mRNA, maximizing the overall release efficiency.

[0057] 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 injury to other organs caused by the leakage of drug proteins.

[0058] In a preferred embodiment of the present invention, the drug proteins are the genes (SEQ ID No. 23) of pyroptosis protein GSDMD-N (the N-terminal fragment of GSDMD), the protein sequence of GSDMD-N (SEQ ID No. 24), the genes (SEQ ID No. 25) of GSDME-N (the N-terminal fragment of GSDME), and the protein sequence of GSDME-N (SEQ ID No. 26).

[0059] In a preferred embodiment of the present invention, the pharmaceutical protein may also be the gene (SEQ ID No. 44) of pyroptosis protein GSDMA-N (N-terminal fragment of GSDMA) and the protein sequence of GSDMA-N (SEQ ID No. 45), the gene (SEQ ID No. 46) of GSDMB-N (N-terminal 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-terminal fragment of GSDMC) and the protein sequence of GSDMC-N (SEQ ID No. 49).

[0060] In a preferred embodiment of the present invention, the expression vector further comprises a hypoxia-specific gene expression cassette, which comprises:

[0061] a) A forward hypoxia promoter, which is a promoter containing an FNR binding site, and the forward hypoxia promoter can be induced to express under hypoxia;

[0062] b) An essential survival gene; and

[0063] c) A reverse hyperoxia promoter, which is a promoter containing FNR and ArcA binding sites, and the reverse hyperoxia promoter can function under the oxygen content conditions of normal organs;

[0064] The essential survival gene is the gene encoding alanine racemase.

[0065] "Polypeptide", "peptide", and "protein" are used interchangeably in the present invention and refer to polymers of amino acid residues. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acids, as well as to polymers of natural amino acids. The terms "polypeptide", "peptide", "amino acid sequence", and "protein" may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0066] In the present invention, "polynucleotide" refers to a macromolecule formed by connecting multiple nucleotides through phosphodiester bonds, where the nucleotides include ribonucleotides and deoxyribonucleotides. The sequence of the polynucleotide of the present invention can be codon-optimized for different host cells (such as Escherichia coli) to improve the expression of polypeptides. The methods for codon optimization are known in the art.

[0067] In order for bacteria to survive only under conditions of low oxygen concentration, three requirements need to be met:

[0068] 1. Control the strength of the upstream promoter and the background leakage expression.

[0069] If the oxygen sensor has partial leaky expression without binding to the upstream promoter, the regulation of oxygen cannot be achieved. As a result, the bacteria will survive at any oxygen concentration; or if the strength of the upstream promoter is too weak, the expression of downstream genes cannot be initiated, leading to the non - survival of the bacteria at any oxygen concentration.

[0070] 2. Select appropriate essential survival genes.

[0071] By selecting essential survival genes, it can be ensured that the bacteria die when the essential survival genes are not expressed, and the survival of the bacteria can be quickly ensured when induced by low oxygen.

[0072] 3. In the case of switching between low oxygen and high oxygen, the upstream promoter can quickly initiate transcription and rapidly achieve the synthetic expression of essential survival genes, ensuring the survival of the modified bacteria. In the case of high oxygen, the expression of essential survival genes will not be initiated, resulting in the death of the bacteria.

[0073] Therefore, in the present invention, the "low - oxygen - specific gene expression cassette" refers to a segment of DNA that can initiate the expression of essential genes under low - oxygen conditions, which contains essential genes controlled by a low - oxygen - inducible promoter and may further contain other regulatory elements required for the expression of the essential genes when necessary.

[0074] In the present invention, the "essential gene" refers to a gene that plays a decisive role in the growth and / or survival of bacteria. Once the bacteria lack this gene or its functional expression product, they cannot survive, divide, and / or grow normally. A typical example of a strain lacking an essential gene or its functional expression product is a auxotrophic strain, which cannot survive, divide, and / or grow normally under in vitro culture conditions or in vivo environments without the presence of specific exogenous supplements. Essential genes usually exist as single copies on the bacterial chromosome.

[0075] From this, the requirements for essential survival genes are as follows:

[0076] 1. It is an essential gene for bacterial reproduction, and its deletion will lead to the rapid death of bacteria.

[0077] 2. The product of this gene does not exist in the normal environment and in the human body, which can ensure that it will not get out of control in the human body environment and can facilitate the cultivation and preparation of bacteria by adding the corresponding expression product of this gene in the normal culture environment.

[0078] 3. This gene needs to be quickly initiated under the regulation of a low - oxygen promoter and can quickly synthesize products to achieve the regulatory function of the host bacteria.

[0079] In the present invention, the essential survival gene is the gene encoding alanine racemase.

[0080] For Gram-negative bacteria such as Escherichia coli and Salmonella, the cell wall is an essential component. The core component in the cell wall is peptidoglycan. When synthesizing peptidoglycan, bacteria require a D-alanine as an important component. Without D-alanine, bacteria cannot synthesize the cell wall and will thus undergo lysis.

[0081] In nature, only L-type amino acids exist. Therefore, Gram-negative bacteria have two genes: the alr gene that biosynthesizes alanine racemase and dadX, which are responsible for converting L-alanine into D-alanine to meet the requirements for cell wall synthesis.

[0082] Studies have found that if both the alr gene and the dadX gene are mutated simultaneously, a lethal mutation of Salmonella can be achieved, and this mutation can be compensated by supplementing D-alanine additionally in the culture medium.

[0083] In our previous study, the YB1 Salmonella used the asd gene as an essential gene for regulation. Compared with the YB1 Salmonella, the present invention selects the alr gene and the dadX gene as essential genes for survival. The alr gene and the dadX gene are functionally homologous genes. Therefore, in the present invention, the gene for alanine racemase can be the alr gene (SEQ ID No.27) from Salmonella and the protein sequence of Salmonella alr (SEQ ID No.28), or the dadX gene (SEQ ID No.29) from Salmonella and the protein sequence of Salmonella dadX (SEQ ID No.30); or the alr or dadX gene of other Gram-negative bacteria or genes with equivalent functions.

[0084] In the present invention, bacteria with the alr gene knocked out are first constructed, and then the other gene dadX is modified, so that the bacteria become defective in both the alr gene and the dadX gene after editing, and at the same time, a forward low-oxygen promoter and a reverse high-oxygen promoter are used to regulate the additional alr or dadX gene.

[0085] The forward low-oxygen promoter described in the present invention is a low-oxygen condition promoter regulated by FNR; the reverse high-oxygen promoter described in the present invention is an antisense promoter negatively regulated by FNR and / or ArcA. The fumarate and nitrate reduction gene fnr is an important gene that regulates the aerobic and anaerobic growth of Salmonella, and this complex regulation system has been widely studied in Escherichia coli and Salmonella. Among them, the DNA-binding protein FNR encoded by the fnr gene senses changes in oxygen and controls the expression of different genes to achieve a switch at the overall metabolic level. Therefore, DNA-binding sequences such as FNR and ArcA have become important ways to control the expression of downstream genes.

[0086] FNR has an oxygen-sensitive [4Fe-4S] 2+ domain that can directly sense oxygen and regulate site-specific DNA binding. In contrast, ArcA senses signals from the aerobic respiratory chain. Therefore, there are two different mechanisms in Gram-negative facultative anaerobes such as Salmonella and Escherichia coli for sensing changes in oxygen concentration. When FNR regulates gene expression under low oxygen, it can be divided into two cases: activation and inhibition.

[0087] For this reason, the present invention uses the forward low-oxygen promoter yhbU and ynfK that activate downstream gene expression under low oxygen by FNR, and the reverse high-oxygen promoter ydcI and cyoA that inhibit downstream gene expression under low oxygen by FNR and / or ArcA.

[0088] Among them, the forward low-oxygen promoters applied in the present invention are as follows:

[0089] 1. The Salmonella yhbU promoter (yhbU-S) (SEQ ID No. 31), which contains the binding site for FNR as "CTGCCTTAAATCAA";

[0090] 2. The Escherichia coli ynfK promoter (ynfK-E) (SEQ ID No. 32), which contains the binding site for FNR as "TTGCGCTATCTCAA";

[0091] Among them, the reverse high-oxygen promoters applied in the present invention are as follows:

[0092] 1. The Salmonella cyoA promoter (cyoA-S) (SEQ ID No. 33), which contains the binding site for FNR as "TTTATTGATAATAA" and the binding site for ArcA as "GTTAAGTA";

[0093] 2. The Salmonella ydcI promoter (ydcI-S) (SEQ ID No. 34), which contains the binding site for FNR as "GTTATCAAAAACAA" and the binding site for ArcA as "GTTAATAA";

[0094] By analyzing the above reverse low-oxygen promoters and forward high-oxygen promoters, we found that the FNR binding sites all conform to the pattern of "TTGATNNNNATCAA", and any base in the TTGAT and ATCAA sequences of the conserved binding site can be replaced, but the total number does not exceed 3, and no 3 consecutive adjacent bases can be replaced; and ArcA conforms to the pattern of its core region "GTTAATTA", and any base in the GTTAATTA sequence of the conserved binding site can be replaced, but the total number does not exceed 2.

[0095] Thus, 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. Any base in the TTGAT and ATCAA sequences of the conserved binding site can be replaced, but the total number of replaced bases does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; the ArcA binding site of the reverse hyperoxia promoter conforms to the pattern of GTTAATTA, and any base therein can be replaced, but the total number of replaced bases does not exceed 2.

[0096] In a more preferred embodiment of the present invention, the forward hypoxia promoters are selected from yhbU and ynfK; the essential genes for survival are selected from alr and dadX; the reverse hyperoxia promoters are selected from cyoA and ydcI.

[0097] In a further preferred embodiment of the present invention, the expression cassette is composed of the forward hypoxia promoter yhbU, the essential gene for survival alr, and the reverse hyperoxia promoter cyoA.

[0098] In a further preferred embodiment of the present invention, the expression cassette is composed of the forward hypoxia promoter yhbU, the essential gene for survival alr, and the reverse hyperoxia promoter ydcI.

[0099] In a further preferred embodiment of the present invention, the expression cassette is composed of the forward hypoxia promoter ynfK, the essential gene for survival dadX, and the reverse hyperoxia promoter cyoA.

[0100] In a further preferred embodiment of the present invention, the expression cassette is composed of the forward hypoxia promoter ynfK, the essential gene for survival dadX, and the reverse hyperoxia promoter ydcI.

[0101] The forward hypoxia promoter disclosed in Patent CN104471057B is pepT. The pepT promoter is not completely regulated by FNR, but is a promoter jointly regulated by CRP-cAMP and FNR. Therefore, half of the FNR binding site in the pepT promoter is the binding site of CRP, and half is the binding site of FNR. The CRP-FNR binding region sequence of the pepT promoter is GTGACCTGACGCAA. The first half GTGA conforms to the first half of the CRP conserved binding site GTGANNNNNNTCAC, and the second half CGCAA conforms to part of the FNR conserved region ATCAA. 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 regarding the forward hypoxia promoter in the present invention.

[0102] The FNR binding region TTGATAATCATTTT of the reverse hyperoxic promoter sodA disclosed in Patent CN104471057B only contains the first half region TTGAT of FNR, and the second half region contains three consecutive substitutions of ATCAA into ATTTT. Therefore, it does not contain a complete FNR binding site. The binding site containing ArcA is TTTAATTA. Compared with the conserved core binding site 5'-GTTAATTA-3' of ArcA, the first G is replaced by T. Therefore, the promoter of sodA only contains a single binding site for ArcA, and its FNR binding site is incomplete, which does not conform to the rules of the reverse hyperoxic promoter in the present invention.

[0103] In summary, the present invention obtains a more efficient and safer hypoxia-specific gene expression cassette by screening reasonable forward hypoxia promoters and reverse hyperoxia promoters, and at the same time cooperating with appropriate essential survival genes.

[0104] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is regulated by the oxygen concentration.

[0105] In order to enable Gram-negative bacteria such as Salmonella to survive in the hypoxic region, 1% oxygen concentration is a very important key point, which represents pathological hypoxia. Because below 1% oxygen concentration is an obvious sign of the tumor hypoxic region, and there is no region with an oxygen concentration below 1% in normal organ tissues. For example, the oxygen concentration in the hypoxic regions of various tumors such as pancreatic cancer, cervical cancer, and prostate cancer is below 0.7%.

[0106] The hypoxia-specific gene expression cassette designed by the present invention enables Gram-negative bacteria such as Salmonella to recognize the tumor hypoxic region through precise regulation of oxygen and proliferate in the tumor hypoxic region. The goal achieved by the present invention is that when the oxygen concentration is below 0.8%, the modified Gram-negative bacteria such as Salmonella can survive and proliferate, and achieve suicidal lysis in the environment of normal oxygen concentration.

[0107] Thus, in a more preferred embodiment of the present invention, the forward hypoxia promoter functions when the oxygen content is below 1%, and does not function when the oxygen content is above 1%; and / or

[0108] The reverse hyperoxia promoter functions when the oxygen content is above 1%, and does not function when the oxygen content is below 1%.

[0109] In a further preferred embodiment of the present invention, the forward hypoxia promoter functions when the oxygen content is below 0.8%, and does not function when the oxygen content is above 0.8%; and / or

[0110] The reverse high-oxygen promoter functions when the oxygen content is higher than 0.8%, and cannot function when the oxygen content is lower than 0.8%.

[0111] In a preferred embodiment of the present invention, among the promoters derived from double phages, one is successively linked to a ribosomal binding site (RBS) (SEQ ID No. 35) and a transmembrane protein gene, and the other is successively linked to an IRES sequence, a kozak sequence, the orf region of a eukaryotic drug, a 3'-UTR region, and successively expressed IRES sequence, kozak sequence, the orf region of a specific phage RNA polymerase gene, a polyA tail, and a T7 terminator sequence.

[0112] In a preferred embodiment of the present invention, the replication origin of the expression vector of the present invention is a high-copy replicon pUC (SEQ ID No. 36), or a low-copy replicon p15A, ColE1, R6K.

[0113] Meanwhile, the expression vector of the present invention does not contain a resistance gene.

[0114] In a preferred embodiment of the present invention, the host bacterium of the expression vector is a Gram-negative bacterium.

[0115] In the present invention, the term "Gram-negative bacterium" refers to a bacterium that does not retain the initial basic dye stain (such as crystal violet) after performing a part of the known procedure of Gram staining. In an exemplary Gram staining, cells are first fixed on a slide by heating and stained with a basic dye (such as crystal violet), which is absorbed by both Gram-negative bacteria and Gram-positive bacteria. Then, the slide is treated with a mordant (such as Gram's iodine solution), which binds to the basic dye (such as crystal violet) and traps it in the cells. Then, the cells are washed with acetone or ethanol and then counterstained with a second dye of a different color (such as safranin). Gram-positive organisms retain the initial purple stain, while Gram-negative organisms are decolorized by the organic washing solvent and thus show the counterstain. Exemplary Gram-negative bacteria include, but are not limited to, bacteria of the genera Escherichia, Shigella, Salmonella, Campylobacter, Neisseria, Haemophilus, Aeromonas, Francisella, Yersinia, Klebsiella, Bordetella, Legionella, Corynebacterium, Citrobacter, Chlamydia, Brucella, Pseudomonas, Helicobacter, and Vibrio.

[0116] Thus, in a more preferred embodiment of the present invention, the Gram-negative bacteria are selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simonsiella, Enterobacter ludwigii, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Bordetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, Vibrio parahaemolyticus, Vibrio cholerae, Yersinia pestis, Catococcus, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolytica, Salmonella enterica, Salmonella bongori, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori.

[0117] In the present invention, the term "living bacteria" refers to a strain with vital, active nutritional and metabolic characteristics and capable of exercising its own biological functions. Living bacteria may include bacterial biomass produced during the strain's metabolic process.

[0118] In a more preferred embodiment of the present invention, the Gram-negative bacteria are Salmonella.

[0119] On the other hand, the present invention provides a method for constructing a constitutive eukaryotic drug mRNA / DNA hybrid dual-delivery cytoplasmic expression system containing a dual-phage-derived promoter, comprising the following steps:

[0120] 1. Sequentially connect the phage-derived promoter, ribosome binding site, and membrane-breaking protein gene of the present invention to construct a membrane-breaking protein gene expression unit;

[0121] 2. Sequentially connect the phage-derived promoter IRES sequence, kozak sequence, orf region of the eukaryotic drug, 3'-UTR region, and sequentially expressed IRES sequence, kozak sequence, orf region of the specific phage RNA polymerase gene, polyA tail, and T7 terminator sequence to construct a drug eukaryotic protein gene transcription unit and a self-enhancing unit in the eukaryotic cytoplasm.

[0122] On the other hand, the present invention provides a method for controlling the expression of drug mRNA in prokaryotic cells using the expression vector of the present invention, which comprises the following steps:

[0123] 1. Prepare the expression vector of the present invention;

[0124] 2. Transform the constitutive promoter that controls the expression of the phage RNA polymerase gene into the chromosome of Gram-negative bacteria;

[0125] 3. Transform the expression vector of the present invention into Gram-negative bacteria.

[0126] When the expression vector of the present invention is transferred into Gram-negative bacteria (such as Salmonella typhimurium) as therapeutic bacteria, the efficient expression of the drug protein gene and the membrane-breaking protein gene controlled by the specific phage RNA polymerase integrated into the bacterial chromosome and the promoter derived from the double phage will lead to an increase in the toxicity of the bacteria. Therefore, in order to achieve the safety of the therapeutic bacteria, the bacteria integrated with the expression vector of the present invention need to be modified by one or more attenuation methods. The attenuation methods include, but are not limited to, the hypoxia-specific gene expression cassette regulation system, auxotrophy, stress response defect, and virulence island regulation defect, etc.

[0127] Therefore, in a preferred embodiment of the present invention, the Gram-negative bacteria are attenuated Gram-negative bacteria.

[0128] In a more preferred embodiment of the present invention, the attenuated Gram-negative bacteria are Salmonella.

[0129] In a more preferred embodiment of the present invention, the attenuation method is selected from the aroA gene defect of Salmonella and the hypoxia-specific gene expression cassette regulation system.

[0130] On the other hand, the present invention provides a modified Gram-negative bacterium comprising the expression vector of the present invention.

[0131] In a preferred embodiment of the present invention, the Gram-negative bacteria are selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simiduella, Enterobacter ludwigii, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Bordetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, Vibrio parahaemolyticus, Vibrio cholerae, Yersinia pestis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolytica, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori.

[0132] In a more preferred embodiment of the present invention, the Gram-negative bacteria are Salmonella.

[0133] Another aspect of the present invention provides the use of the expression vector described in the present invention or the Gram-negative bacterium described in the present invention in the preparation of anti-tumor drugs.

[0134] In a preferred embodiment of the present invention, the tumor is a solid tumor.

[0135] As used herein, the term "solid tumor" refers to an abnormal mass of tissue that generally does not contain cysts or fluid areas. Solid tumors may be benign (non-cancerous) or malignant (cancerous). Different types of malignant solid tumors are named for the cell types that form them. Examples of malignant solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (blood cancers) generally do not form malignant solid tumors. Malignant solid tumors include, but are not limited to, abnormal cell masses that may originate from different tissue types such as the liver, colon, colorectum, skin, breast, pancreas, cervix, corpus uteri, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testis, thyroid, or lung, etc. Thus, malignant solid tumors include malignant solid liver tumors, colon tumors, colorectal tumors, skin tumors, breast tumors, pancreatic tumors, cervical tumors, corpus uteri 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.

[0136] Thus, in a more preferred embodiment of the present invention, the solid tumors are selected from tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin lymphoma, Hodgkin lymphoma), intestine (colon cancer, rectal cancer), pelvis (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer, and bladder.

[0137] In a further preferred embodiment of the present invention, the solid tumors are breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma, and bladder cancer.

[0138] In the present invention, for treatment purposes, the term "subject" preferably refers to a subject in need of treatment for a target pathological condition such as a tumor. For prevention purposes, 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 particular embodiment of the present invention, the subject is a human.

[0139] As used herein, "treatment" is a process for obtaining a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are 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 malignancy, alleviating the symptoms caused by a malignancy, improving the quality of life of a subject having a malignancy, reducing the dosage of other medications required to treat a malignancy, delaying the progression of a malignancy, curing a malignancy, and / or prolonging the survival of a patient having a malignancy.

[0140] 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 outcomes. For prophylactic use, beneficial or desired outcomes include eliminating or reducing the risk of a disease, reducing the severity of a disease or delaying the onset of a disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during the development of the disease. For therapeutic use, beneficial or desired outcomes include, for example, alleviating one or more symptoms of a disease (such as a tumor), reducing the dosage of other medications required to treat a 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%, more preferably at least about 80% relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy against human tumors. Alternatively, it can also 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 is capable of reducing the size of a tumor or otherwise alleviating the symptoms of a subject. Those skilled in the art can determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0141] As used herein, "pharmaceutically acceptable carrier" includes any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the immune system of the subject, 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 encapsulated in a material to protect the bacteria from the action 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 injectable solutions or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except to the extent that they are incompatible with the active compound, may be present in the pharmaceutical compositions of the present invention.

[0142] Accordingly, in another aspect, 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.

[0143] In a preferred embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0144] 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.

[0145] In a preferred embodiment of the present invention, the pharmaceutical composition is used for treating solid tumors. The bacteria, drugs or pharmaceutical compositions of the present invention are 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.

[0146] In a preferred embodiment of the present invention, the bacteria, drugs or pharmaceutical compositions of the present invention can be formulated into forms 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.

[0147] The dosage form of the drug or pharmaceutical composition of the present invention may be in the form of a solution, emulsion, freeze-dried preparation or suspension; for oral administration, the dosage form may be in the form of tablets or capsules; for intranasal dosage forms, the dosage form may be in the form of powders, nasal drops or aerosols; for topical application, the dosage form may be an aqueous solution, suspension, ointment, cream or gel; for rectal or vaginal administration, the dosage form may be a suppository, enema or delivered as part of an endoscopic or colonoscopy procedure.

[0148] The bacteria, drugs or pharmaceutical compositions of the present invention can be manufactured by methods well known in the art, such as microbial growth in a fermenter, followed by centrifugal concentration and washing, filtration or dialysis, conventional granulation, mixing, dissolution, encapsulation, lyophilization or emulsification processes and other methods. The bacteria, drugs or pharmaceutical compositions of the present invention can be produced in various forms, including granules, precipitates or microparticles, powders, including freeze-dried, rotary-dried or spray-dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. The formulation may optionally contain stabilizers, pH regulators, surfactants, bioavailability regulators and combinations thereof.

[0149] The bacteria, drugs or pharmaceutical compositions of the present invention can be administered alone or in combination with other compounds or compositions in the presence of a carrier. In a preferred embodiment of the present invention, the bacteria, drugs or pharmaceutical compositions can be administered in combination with other malignant tumor therapies (including but not limited to, radiotherapy, chemotherapy and surgery). The bacteria, drugs or pharmaceutical compositions can be used as adjuvants in such therapies.

[0150] By adopting the above technical solutions, the present invention has obtained the following beneficial effects:

[0151] 1. By combining a phage-derived promoter with a specific phage RNA polymerase gene, the present invention enables the two to cooperate, and through a plasmid-based expression platform, stable and efficient expression of drug proteins in the tumor region is achieved.

[0152] 2. By combining a phage-derived promoter with a membrane-breaking protein gene and a eukaryotic drug gene mRNA / DNA hybrid vector, the present invention enables the three to cooperate, stably and efficiently deliver drugs, achieve a true cytoplasmic expression system, and thus achieve efficient killing of tumor cells.

[0153] 3. By reasonably designing a hypoxia-specific gene expression cassette, the present invention enables the modified bacteria to have a low overall mutation rate, achieve specific distribution inside tumors, and have a faster clearance rate in normal organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0154] Figure 1Agarose gel electrophoresis identification of the forward hypoxia promoter yhbU-S and ynfK-S clone verification diagram.

[0155] Figure 2 Agarose gel electrophoresis identification of the essential gene library verification amplification diagram.

[0156] Figure 3 Agarose gel electrophoresis identification of the reverse hyperoxia promoter cyoA-S and ydcI-S clone verification diagram.

[0157] Figure 4 Schematic diagram of the hypoxia-specific gene expression cassette.

[0158] Figure 5 Schematic diagram of the lambda RED recombinase and CRE recombinase systems.

[0159] Figure 6 Identification diagram of the knockout experiment of the Salmonella aroA gene.

[0160] (A) Agarose gel electrophoresis identification of the PCR amplification product of the aroA gene knockout fragment verification diagram;

[0161] (B) Agarose gel electrophoresis identification of the PCR amplification product of the target fragment of strain SWT003 verification diagram.

[0162] Figure 7 Identification diagram of the knockout experiment of the Salmonella alr.

[0163] (A) Agarose gel electrophoresis identification of the PCR amplification product of the alr gene knockout fragment verification diagram;

[0164] (B) Agarose gel electrophoresis identification of the PCR amplification product of the target fragment of strain SWT004 verification diagram.

[0165] Figure 8 Agarose gel electrophoresis identification of strains SWT1001 and SWT1005 after insertion of the hypoxia-specific gene expression cassette (alr is an essential gene).

[0166] Figure 9 Agarose gel electrophoresis identification of the PCR amplification product of SWT007.

[0167] Figure 10 Agarose gel electrophoresis identification of strains SWT2009 and SWT2013 after insertion of the hypoxia-specific gene expression cassette (dadX is an essential gene).

[0168] Figure 11 Results diagram of the oxygen adaptability verification test of Salmonella strain SWT1001.

[0169] (A) Cultured under anaerobic conditions on an LB plate without D-alanine;

[0170] (B) Cultured under aerobic conditions on an LB plate without D-alanine.

[0171] Figure 12 Results graph of the oxygen adaptability verification test for Salmonella strain SWT1005.

[0172] (A) Cultured under anaerobic conditions on an LB plate without D-alanine;

[0173] (B) Cultured under aerobic conditions on an LB plate without D-alanine.

[0174] Figure 13 Oxygen adaptability verification graph for strains SWT2009 and SWT2013.

[0175] (A) For SWT2009 on an LB plate without D-alanine, anaerobic culture on the top and aerobic culture on the bottom;

[0176] (B) For SWT2013 on an LB plate without D-alanine, anaerobic culture on the top and aerobic culture on the bottom.

[0177] Figure 14 Results graph of the oxygen adaptability verification test for each Salmonella strain when the oxygen concentration is below 0.8%.

[0178] (A) SWT1001 cultured on an LB plate;

[0179] (B) SWT1005 cultured on an LB plate;

[0180] (C) SWT2009 cultured on an LB plate;

[0181] (D) SWT2013 cultured on an LB plate.

[0182] Figure 15 Schematic diagram of the chloramphenicol resistance gene, lacUV5, and T7 RNA polymerase gene cassette pPRO005.

[0183] Figure 16 Restriction enzyme digestion identification graph of plasmid pPRO005 (the arrow indicates the excised T7 RNA polymerase gene cassette fragment).

[0184] Figure 17 Identification graph of the recombination of the lacUV5-controlled constitutive expression cassette of T7 RNA polymerase into the Salmonella chromosome.

[0185] (A) PCR amplification fragment for recombination (primers SWTO102 and SWTO103);

[0186] (B) PCR identification diagrams of strains SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P were constructed.

[0187] Figure 18 Schematic diagram of the construction and function of plasmid vector pEU011.

[0188] (A) Construction diagram of plasmid vector pEU011;

[0189] (B) Schematic diagram of the process of plasmid vector pEU011 exerting its function.

[0190] Figure 19 PCR identification diagram of plasmid vector pEU011.

[0191] Figure 20 Schematic diagram of the construction and function of plasmid vector pEU010.

[0192] (A) Construction diagram of plasmid vector pEU010;

[0193] (B) Schematic diagram of the process of plasmid vector pEU010 exerting its function.

[0194] Figure 21 PCR identification diagram of plasmid vector pEU010.

[0195] Figure 22 Schematic diagram of the construction and function of plasmid vector pIKDE-EGFP.

[0196] (A) Construction diagram of plasmid vector pIKDE-EGFP;

[0197] (B) Schematic diagram of the process of plasmid vector pIKDE-EGFP exerting its function.

[0198] Figure 23 Observation of the expression intensity of green fluorescence after transfection of plasmids pEU010, pEU011, and pIKDE-EGFP into BSR-T7 / 5 cells with stable expression of T7 polymerase under a fluorescence microscope.

[0199] (A) BSR-T7 / 5 cell control, the upper is the white light image and the lower is the fluorescence image;

[0200] (B) BSR-T7 / 5 cells transfected with pEU010, the upper is the white light image and the lower is the fluorescence image;

[0201] (C) BSR-T7 / 5 cells transfected with pEU011, the upper is the white light image and the lower is the fluorescence image;

[0202] (D) BSR-T7 / 5 cells were transfected with pIKDE-EGFP. The upper panel shows the white light image, and the lower panel shows the fluorescence image.

[0203] Figure 24 Flow cytometry was used to analyze the proportion of green fluorescent cells after transfection of pEU010, pEU011, and pIKDE-EGFP plasmids into BSR-T7 / 5 cells stably expressing T7 polymerase.

[0204] (A) BSR-T7 / 5 cells were transfected with pEU010;

[0205] (B) BSR-T7 / 5 cells were transfected with pEU011;

[0206] (C) BSR-T7 / 5 cells were transfected with pIKDE-EGFP.

[0207] Figure 25 Schematic diagram of the construction of plasmid pSWT004 for the construction of YB1-like Salmonella SWT005.

[0208] Figure 26 Verification diagram of the restriction enzyme digestion products of plasmid pSWT004 by agarose gel electrophoresis.

[0209] Figure 27 Verification diagram of the PCR amplification products of the cm-pepT-asd-sodA fragment by agarose gel electrophoresis.

[0210] Figure 28 Verification diagram of the PCR amplification products of Salmonella SWT005 by agarose gel electrophoresis.

[0211] Figure 29 Results diagram of the oxygen adaptability verification test of YB1-like Salmonella SWT005.

[0212] (A) Cultured under anaerobic conditions on an LB plate without D-alanine;

[0213] (B) Cultured under aerobic conditions on an LB plate without D-alanine.

[0214] Figure 30 PCR identification diagram of strain SWT008.

[0215] Figure 31 Schematic diagram of plasmid vector pPRO013.

[0216] Figure 32 PCR identification diagram of strain SWT5025.

[0217] Figure 33 Compare the growth ability of strains SWT5015, SWT5015C, and SWT5025 in the medium.

[0218] (A) Growth curves of strains SWT5015, SWT5015C and SWT5025 in liquid LB medium;

[0219] (B) Counting analysis of strains SWT5015, SWT5015C and SWT5025 on solid LB plates under the same OD conditions.

[0220] Figure 34 Identification of mRNA transcription of EGFP in strains SWT5015, SWT5015C and SWT5025.

[0221] (A) Identification of total RNA extracted from control strain SWT1005 and strains SWT5015, SWT5015C and SWT5025 by agarose gel electrophoresis;

[0222] (B) Amplification of EGFP RNA from control strain SWT1005 and strains SWT5015, SWT5015C and SWT5025 by reverse transcription qPCR and non-reverse transcription qPCR respectively.

[0223] Figure 35 Western Blot identification of the expression of T7 RNA polymerase and LLO in strains SWT5025, SWT5015C and SWT5025.

[0224] (A) Expression of T7 RNA polymerase in strains SWT5025, SWT5015C and SWT5025 under the conditions of LB medium and N-salts medium;

[0225] (B) Expression of LLO in strains SWT5025, SWT5015C and SWT5025 under the conditions of LB medium and N-salts medium.

[0226] Figure 36 Comparison of the expression efficiency of EGFP after strains SWT5015C, SWT5015 and SWT5025 invade cancer cells.

[0227] (A) Observation of EGFP expression in invaded cells under a fluorescence microscope;

[0228] (B) Analysis of the fluorescence signal of EGFP by an enzyme-labeled instrument.

[0229] Figure 37 PCR identification diagrams of plasmid vectors pEU013 and pEU014.

[0230] Figure 38qPCR identification of the transcription of the target gene mRNA in SWT5115, SWT5119, SWT5215, and SWT5219. The Cq value represents the amplification threshold.

[0231] (A) Reverse transcription qPCR and non-reverse transcription qPCR were used to amplify GSDMD-N mRNA in the control strain SWT1005 and SWT5115, respectively.

[0232] (B) Reverse transcription qPCR and non-reverse transcription qPCR were used to amplify GSDME-N mRNA in the control strain SWT1005 and SWT5215, respectively.

[0233] (C) Reverse transcription qPCR and non-reverse transcription qPCR were used to amplify GSDMD-N mRNA in the control strain SWT1005 and SWT5119, respectively.

[0234] (D) Reverse transcription qPCR and non-reverse transcription qPCR were used to amplify GSDME-N mRNA in the control strain SWT1005 and SWT5219, respectively.

[0235] Figure 39 Microscopic images of the killing of cells caused by pyroptosis after co-culturing the control strain SWT1005, SWT5115, and SWT5215 with cancer cells under anaerobic conditions.

[0236] (A) EMT6;

[0237] (B) MF C ;

[0238] (C) RM-1.

[0239] Figure 40 Microscopic images of the killing of cells caused by pyroptosis after co-culturing the control strain SWT1005, SWT5115, and SWT5215 with cancer cells under anaerobic conditions.

[0240] (D) SCC7;

[0241] (E) CT26;

[0242] (F) MB49.

[0243] Figure 41 Microscopic images of the killing of cells caused by pyroptosis after co-culturing the control strain SWT1005, SWT5115, and SWT5215 with cancer cells under anaerobic conditions.

[0244] (G) A549;

[0245] (H) Neuro-2a;

[0246] (I) ID8。

[0247] Figure 42 Microscopic images of the killing effect on cells caused by pyroptosis after co - culturing strain SWT1005 as a control, SWT5115, and SWT5215 with cancer cells under anaerobic conditions.

[0248] (J) Hepa1 - 6;

[0249] (K) Renca;

[0250] (L) Pan02.

[0251] Figure 43 Microscopic images of the killing effect on cells caused by pyroptosis after co - culturing strain SWT1005 as a control, SWT5115, and SWT5215 with cancer cells under anaerobic conditions.

[0252] (M) SK - MEL - 5;

[0253] (N) K7M2.

[0254] Figure 44 Graph showing the killing effect of control strain SWT1005 and expression vector strains SWT5115 and SWT521 on EMT6, K7M2, Hepa1 - 6, A549, SK - MEL - 5, Renca, MFC, Pan02, RM - 1, CT26, ID8, Neuro - 2a, SCC7, and MB49 cancer cells as determined by CCK8 assay.

[0255] Figure 45 Microscopic images of the killing effect on cells caused by pyroptosis after co - culturing strain SWT2009 as a control, SWT5119, and SWT5219 with cancer cells under anaerobic conditions.

[0256] (A) EMT6;

[0257] (B) MF C ;

[0258] (C) RM - 1.

[0259] Figure 46 Microscopic images of the killing effect on cells caused by pyroptosis after co - culturing strain SWT2009 as a control, SWT5119, and SWT5219 with cancer cells under anaerobic conditions.

[0260] (D) SCC7;

[0261] (E) CT26;

[0262] (F) MB49。

[0263] Figure 47 The strain SWT2009 was used as a control, and after SWT5119 and SWT5219 were co-cultured with cancer cells under anaerobic conditions, respectively, the microscopic images of the killing of cells caused by pyroptosis.

[0264] (G) A549;

[0265] (H) Neuro-2a;

[0266] (I) ID8.

[0267] Figure 48 The strain SWT2009 was used as a control, and after SWT5119 and SWT5219 were co-cultured with cancer cells under anaerobic conditions, respectively, the microscopic images of the killing of cells caused by pyroptosis.

[0268] (J) Hepa1-6;

[0269] (K) Renca;

[0270] (L) Pan02.

[0271] Figure 49 The strain SWT2009 was used as a control, and after SWT5119 and SWT5219 were co-cultured with cancer cells under anaerobic conditions, respectively, the microscopic images of the killing of cells caused by pyroptosis.

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

[0273] (N) K7M2.

[0274] Figure 50 The CCK8 assay results showing the killing effects of the control strain SWT2009 and the expression vector strains SWT5119 and SWT5219 on EMT6, K7M2, A549, SK-MEL-5, MFC, and SCC7 cancer cells.

[0275] Figure 51 Identification of the inhibitory effects of the expression vector strains SWT5115, SWT5215 and the control strain SWT1005 in different mouse tumor models (vehicle is the blank control, without drug treatment).

[0276] (A) EMT6;

[0277] (B) K7M2;

[0278] (C) Hepa1-6;

[0279] (D) A549.

[0280] Figure 52 Identification diagrams of the inhibitory effects of expression vector strains SWT5115, SWT5215 and control strain SWT1005 in different mouse tumor models (vehicle is the blank control, without drug treatment).

[0281] (A) B16F10;

[0282] (B) Renca;

[0283] (C) MFC;

[0284] (D) Pan02.

[0285] Figure 53 Identification diagrams of the inhibitory effects of expression vector strains SWT5115, SWT5215 and control strain SWT1005 in different mouse tumor models (vehicle is the blank control, without drug treatment).

[0286] (A) RM-1;

[0287] (B) CT26;

[0288] (C) ID8;

[0289] (D) Neuro-2a.

[0290] Figure 54 Identification diagrams of the inhibitory effects of expression vector strains SWT5115, SWT5215 and control strain SWT1005 in different mouse tumor models (vehicle is the blank control, without drug treatment).

[0291] (A) SCC7;

[0292] (B) MB49. Detailed implementation manners

[0293] The present application will be further described in detail below with reference to the accompanying drawings and embodiments. Through these descriptions, the features and advantages of the present application will become clearer and more definite.

[0294] Example 1: Construction of a hypoxia-specific gene expression cassette with alr gene and dadX gene as essential genes for survival

[0295] In this embodiment, the forward hypoxia promoter uses the Salmonella yhbU (yhbU-S) (SEQ ID No. 31), the essential gene for survival uses the alr gene of Salmonella (SEQ ID No. 27), the reverse hyperoxia promoters respectively use the Salmonella cyoA (cyoA-S) (SEQ ID No. 33), the Salmonella ydcI (ydcI-S) (SEQ ID No. 34), and the forward hypoxia promoter uses the Escherichia coli ynfK (ynfK-E) (SEQ ID No. 32), the essential gene for survival uses the dadX gene of Salmonella (SEQ ID No. 29), and the reverse hyperoxia promoters respectively use the Salmonella cyoA (cyoA-S) (SEQ ID No. 33), the Salmonella ydcI (ydcI-S) (SEQ ID No. 34) to construct a hypoxia-specific gene expression cassette.

[0296] The combination forms of the hypoxia-specific gene expression cassettes are shown in Table 1.

[0297] Table 1 Combination table of forward hypoxia promoters and reverse hyperoxia promoters (where the essential gene for survival is the alr gene or dadX gene of Salmonella)

[0298]

[0299] (I) Construction of a forward hypoxia promoter clone library

[0300] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from the China Center of Industrial Culture Collection of Microorganisms, CICC) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) 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, with the OD 600 reading between 2 and 3;

[0301] 2. Use a pipette tip to aspirate 2 μl of the corresponding strain culture solution and mix it into the corresponding primer and high-fidelity PCR amplification enzyme (purchased from TAKARA) system, and then mix and place it in a PCR amplification device.

[0302] The amplification program is 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, cycle 30 times; 72°C for 10 minutes; 4°C for 5 minutes;

[0303] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0304] 4. Mix the recovered product with the endonuclease (purchased from NEB) digestion system and incubate it at 37°C for 1 hour;

[0305] 5. Purify and recover the digested products using a DNA gel extraction kit.

[0306] The primers and restriction enzymes used for different forward hypoxia promoters are as follows:

[0307] 1. yhbU-S (SEQ ID No. 31): Amplify strain SWT001 using primers SWTO19 and 20, double-digest the product with NotI and HindIII, and recover it.

[0308] 2. ynfK-E (SEQ ID No. 32): Amplify Escherichia coli strain DH10B using primers SWTO21 and 22, double-digest the product with NotI and HindIII, and recover it.

[0309] The results of verifying the forward hypoxia promoter clone library by agarose gel electrophoresis are shown in the appendix Figure 1 as follows.

[0310] (II) Construction of the essential gene library

[0311] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Center of Industrial Culture Collection of Microorganisms) 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.

[0312] 2. Use a pipette tip to aspirate 2 μl of the corresponding strain culture solution and mix it into the corresponding primer and high-fidelity PCR amplification enzyme system, and then mix and place it in a PCR amplification device.

[0313] The amplification program is as follows: 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, for 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes.

[0314] 3. Purify and recover the amplified PCR products using a DNA gel extraction kit.

[0315] 4. Mix the recovered products with the digestion system and incubate at 37°C for 1 hour.

[0316] 5. Purify and recover the digested products using a DNA gel extraction kit.

[0317] The essential genes from different sources are as follows:

[0318] 1. Salmonella alr gene (SEQ ID No. 27): Amplify strain SWT001 using primers SWTO35 and 36, double-digest the product with HindIII and XhoI, and recover it.

[0319] 2. Salmonella dadX gene (SEQ ID No.29): Amplify strain SWT001 with primers SWTO39 and 40, double-digest the product with HindIII and XhoI, and recover it;

[0320] The results of verifying the essential gene library by agarose gel electrophoresis are shown in the appendix Figure 2 as follows.

[0321] (III) Construction of the reverse hyperoxic promoter library

[0322] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Center for Industrial Culture Collection of Microorganisms) 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;

[0323] 2. Use a pipette tip to aspirate 2 μl of the corresponding strain culture solution and mix it into the corresponding primer and high-fidelity PCR amplification enzyme system, and then mix and place it in a PCR amplification device.

[0324] The amplification program is 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, cycle 30 times; 72°C for 10 minutes; 4°C for 5 minutes;

[0325] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0326] 4. Mix the recovered product with the digestion system and incubate it at 37°C for 1 hour;

[0327] 5. Purify and recover the digested product through a DNA gel recovery system.

[0328] The primers and restriction enzymes used for different reverse hyperoxic promoters are as follows:

[0329] 1. cyoA-S (SEQ ID No.33): Amplify strain SWT001 with primers SWTO43 and 44, double-digest the product with XhoI and PstI, and recover it;

[0330] 2. ydcI-S (SEQ ID No.34): Amplify strain SWT001 with primers SWTO51 and 52, double-digest the product with XhoI and PstI, and recover it;

[0331] The results of verifying the reverse hyperoxic promoter clone library by agarose gel electrophoresis are shown in the appendix Figure 3 as follows.

[0332] (IV) Construction of the forward hypoxic promoter, essential gene, and reverse hyperoxic promoter combination library

[0333] The composition of the hypoxia-specific gene expression cassette is shown in the attached Figure 4 As shown, it is composed of a forward hypoxia promoter, a survival essential gene and a reverse hyperoxia promoter in sequence.

[0334] 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.

[0335] 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.

[0336] (V) Construction of the strain corresponding to the present invention

[0337] 1. Wild-type Salmonella Typhimurium (SWT001)

[0338] Wild-type Salmonella typhimurium (SWT001) was purchased from China Industrial Microbiology Culture Collection Center (CICC).

[0339] 2. Construction of Salmonella (SWT002) containing temperature-inducible lambda-RED recombinase and loxp-CRE enzyme systems

[0340] 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.

[0341] Plasmid pSWT001 is attached Figure 5As shown, it 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 technique is that it precisely targets the target region of the bacterial chromosome without inducing additional mutations. The outermost part of the recombinant double-stranded DNA fragment includes the homology arm sequence of the position where recombination is required, and inside it are loxp sequences in the same direction on both sides, totaling 34bp. The site of the double loxp in the same direction is the chloramphenicol resistance gene, which is used to screen recombinant bacteria. After successful recombination, the CRE enzyme system carried on the pSWT001 plasmid (also controlled by the CI857 temperature control 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 operation can achieve continuous knockout and knock-in of genes.

[0342] The specific steps are as follows:

[0343] (1) Streak the strain SWT001 on an LB plate and culture it in a 37°C incubator overnight;

[0344] (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;

[0345] (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;

[0346] (4) Wash the cells three times with sterilized purified water;

[0347] (5) The recovered cells were mixed with 10 ng of plasmid pSWT001 and electroporated at a voltage of 1.8 kV;

[0348] (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.

[0349] 3. Construction of attenuated Salmonella with aroA gene knockout (SWT003)

[0350] (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.

[0351] (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.

[0352] (3) Wash the cells three times with sterilized purified water.

[0353] (4) Prepare PCR products of SWTO1 and SWTO2.

[0354] 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.

[0355] 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.

[0356] The amplified PCR products were verified by agarose gel electrophoresis for the PCR amplification products of SWTO1 and SWTO2 of the aroA gene knockout fragment. The verification results are shown in the attached Figure 6 As shown in A.

[0357] 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.

[0358] (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.

[0359] (6) Spread the electrotransformed bacteria on a plate containing 25 μg / ml chloramphenicol and incubate overnight in a constant temperature incubator at 32 °C until single clone colonies grow.

[0360] (7) Identify positive clones by colony PCR and use SWTO4 and SWTO6, SWTO3 and SWTO5 to identify the insertion of the chloramphenicol resistance gene.

[0361] Verify the PCR amplification product of the target fragment of strain SWT003 by agarose gel electrophoresis, and the verification results are as shown in Appendix Figure 6 B.

[0362] (8) Inoculate the positive monoclonal into 5 ml of LB medium and culture it in a constant temperature shaker at 37 °C and 220 rpm for 16 hours. Let the CRE enzyme act to remove the chloramphenicol resistance gene.

[0363] 4. Construction of the attenuated bacterium (SWT004) with the alr gene knocked out

[0364] (1) Pick a single clone of SWT003 and inoculate it into 5 ml of LB liquid medium, and place it in a constant temperature shaker at 32 °C and 220 rpm for 16 hours.

[0365] (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. Then place the culture flask in a 42 °C water bath, shake it for 15 minutes, and then place it on ice for 1 hour.

[0366] (3) Wash the bacteria 3 times with sterilized pure water.

[0367] (4) Prepare the PCR products of SWTO70 and SWTO71.

[0368] Mix the primers SWTO70 and SWTO71 with the plasmid pSWT002 and the high-fidelity PCR amplification enzyme system and put them into the PCR amplification equipment.

[0369] The amplification program is 95 °C for 2 minutes; 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 60 seconds, cycle 30 times; 72 °C for 10 minutes; 4 °C for 5 minutes.

[0370] Purify and recover the amplified PCR products through a DNA gel recovery system, and measure the DNA concentration and purity of the recovered PCR products by nanodrop.

[0371] (5) Mix the recycled bacterial cells with the PCR products of 100 ng of SWTO70 and SWTO71, and perform electrotransformation at a voltage of 1.8 kV.

[0372] Verify the PCR amplification products of SWTO70 and SWTO71 of the alr gene knockout fragment by agarose gel electrophoresis. The verification results are as shown in Figure 7 Appendix A.

[0373] (6) Spread the electrotransformed bacterial cells on a plate containing 25 μg / ml chloramphenicol, and incubate overnight in a constant temperature incubator at 32 °C until single clone colonies grow.

[0374] (7) Identify positive clones by colony PCR, and use SWTO72 and SWTO5, SWTO6 and SWTO73 to identify the insertion of the chloramphenicol resistance gene.

[0375] Verify the PCR amplification products of the target fragment of strain SWT004 by agarose gel electrophoresis. The verification results are as shown in Figure 7 Appendix B (the primers used are SWTO72, SWTO5, SWTO6, SWTO73).

[0376] (8) Inoculate the positive monoclonal into 5 ml of LB medium, and culture it in a constant temperature shaker at 37 °C and 220 rpm for 16 hours. Let the CRE enzyme act to remove the chloramphenicol resistance gene.

[0377] Example 2: Integrate the hypoxia-specific gene expression cassette into the dadX gene locus (disrupt its function) on the chromosome of strain SWT004

[0378] 1. Pick a single clone of the attenuated bacterium SWT004 with the alr gene knockout and inoculate it into 5 ml of fresh LB liquid medium, and place it in a constant temperature shaker at 32 °C and 220 rpm for 16 hours.

[0379] 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. Then place the culture flask in a 42 °C water bath, shake it for 15 minutes, and then place it on ice and let it stand for 1 hour.

[0380] 3. Wash the bacterial cells 3 times with sterilized pure water.

[0381] 4. Prepare the PCR products for constructing the strain containing the hypoxia-specific expression cassette. Mix the primers SWTO78 and SWTO79 with the hypoxia-specific gene expression cassette plasmids pOL1001, pOL1005, and the high-fidelity PCR amplification enzyme system and put them into the PCR amplification equipment.

[0382] The amplification program was 2 minutes at 95°C; 30 seconds at 95°C, 30 seconds at 60°C, 120 seconds at 72°C, for 30 cycles; 10 minutes at 72°C; 5 minutes at 4°C.

[0383] The amplified PCR products were purified and recovered by a DNA gel recovery system, and the concentration and purity of the DNA in the recovered PCR products were determined by nanodrop.

[0384] 5. Mix the recovered bacteria with the PCR products of 100 ng and perform electrotransformation at a voltage of 1.8 kV.

[0385] 6. Spread the electrotransformed bacteria on plates containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and incubate overnight in a constant temperature incubator at 32°C until single colony clones grow. The generated strains were SWT1001 and SWT1005 respectively.

[0386] 7. Inoculate the monoclonal into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and culture it in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The chloramphenicol resistance gene was removed by the action of CRE enzyme.

[0387] 8. Use primers SWTO61, 20; SWTO75, 20 to identify the clones 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 as shown in the appendix Figure 8 as follows.

[0388] Example 3: Construction of a hypoxia-specific gene expression cassette with the dadX gene as an essential gene for survival

[0389] Since, as alanine racemase, there are two isozyme genes on the genomes of bacteria such as Salmonella and Escherichia coli, namely the alr gene and the dadX gene. To verify the effect, correspondingly, the essential genes for survival of the above-mentioned partial hypoxia-specific gene expression cassettes were replaced with the corresponding homologous genes dadX genes pOL2009 and pOL2013.

[0390] 1. Construction of an attenuated bacterium with the dadX gene knocked out (SWT007)

[0391] (1) Pick a single colony of SWT003 and inoculate it into 5 ml of LB liquid medium, and place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0392] (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. Then, place the culture flask into a 42°C water bath, shake and culture for 15 minutes, and then place it on ice and let it stand for 1 hour.

[0393] (3) Wash the bacterial cells 3 times with sterilized pure water.

[0394] (4) Prepare the PCR products of SWTO78 and SWTO79.

[0395] Mix the primers SWTO78 and SWTO79 with the plasmid pSWT002 (described above, plasmid description), and the high-fidelity PCR amplification enzyme system, and place them into a PCR amplification device.

[0396] The amplification program is: 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, for 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes.

[0397] Purify and recover the amplified PCR products through a DNA gel recovery system, and measure the DNA concentration and purity of the recovered PCR products by nanodrop.

[0398] (5) Mix the recovered bacterial cells with 100 ng of the PCR products of SWTO78 and SWTO79, and perform electrotransformation at a voltage of 1.8 kV.

[0399] (6) Spread the electrotransformed bacterial cells on a plate containing 25 μg / ml chloramphenicol, and incubate overnight in a 32°C constant temperature incubator until single clone colonies grow.

[0400] (7) Identify positive clones by colony PCR, and use SWTO59, SWTO5, SWTO6, and SWTO87 to identify the insertion of the chloramphenicol resistance gene, as shown in the appendix Figure 9 as follows.

[0401] (8) Inoculate the positive monoclonal into 5 ml of LB medium, and culture it in a 37°C constant temperature shaker at 220 rpm for 16 hours. Let the CRE enzyme act to remove the chloramphenicol resistance gene.

[0402] 2. Construction of a defective attenuated bacterium containing a hypoxia-specific gene expression cassette and with the essential gene for survival being the Salmonella dadX gene

[0403] (1) Pick a single clone of the attenuated bacterium SWT007 with the dadX gene knocked out and inoculate it into 5 ml of LB liquid medium, and place it in a 32°C, 220 rpm constant temperature shaker and culture for 16 hours.

[0404] (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. Then place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place it on ice and let it stand for 1 hour.

[0405] (3) Wash the bacterial cells 3 times with sterilized pure water.

[0406] (4) Prepare the PCR products for constructing strains containing hypoxia - specific expression cassettes. Mix the primers SWTO85 and SWTO86 with the hypoxia - specific gene expression cassette plasmids pOL2009, pOL2013, and the high - fidelity PCR amplification enzyme system and place them in a PCR amplification device.

[0407] The amplification program is: 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 120 seconds, cycle 30 times; 72°C for 10 minutes; 4°C for 5 minutes.

[0408] Purify and recover the amplified PCR products through a DNA gel recovery system. Measure the concentration and purity of the DNA of the recovered PCR products by nanodrop.

[0409] (5) Mix the recovered bacterial cells with 100 ng of the PCR products and perform electrotransformation at a voltage of 1.8 kv.

[0410] (6) Spread the electrotransformed bacterial cells on plates containing 25 μg / ml chloramphenicol and 100 μg / ml D - alanine, and incubate them overnight in a 32°C constant - temperature incubator until single - clone colonies grow.

[0411] (7) Inoculate the positive monoclonal into 5 ml of LB medium and culture it in a 37°C constant - temperature shaker at 220 rpm for 16 hours. The chloramphenicol resistance gene is removed by the action of CRE enzyme.

[0412] (8) Use the corresponding primers SWTO98, 22; SWTO99, 22 to identify the clones by colony PCR. The identification results of strains SWT2013(ynfK - E+dadX+ydcI - S) and strain SWT2009(ynfK - E+dadX+cyoA - S) are as shown in the appendix Figure 10 as follows.

[0413] Example 4: Oxygen adaptability verification of Salmonella strains SWT1001, SWT1005, SWT2009, and SWT2013 containing hypoxia - specific gene expression cassettes

[0414] 1. Pick single colonies of strains SWT1001, SWT1005, SWT2009, and SWT2013 containing a hypoxia-specific expression cassette and inoculate them into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and place them in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0415] 2. After culturing, dilute the strains 10-fold and measure the absorbance (OD value) at 600 nm.

[0416] 3. Calculate the volume of the bacterial suspension for 1 OD of bacteria according to the following formula, and supplement deionized water to 1 ml.

[0417]

[0418] 4. Take 10 μl of the above bacterial suspension and spot it onto two LB plates without D-alanine for culturing, label it as "1" and repeat three times, label them as "a", "b", and "c".

[0419] 5. After dilution in a 10-fold gradient, take another 10 μl of the dilution and spot it onto the above medium, label it as "2".

[0420] 6. By analogy with this 10-fold gradient dilution, until it is labeled up to "8".

[0421] 7. Place one of the plates in an anaerobic environment at 37°C for culturing, and place the other plate in an atmospheric environment at 37°C (oxygen concentration of 21%) for culturing.

[0422] The results of the oxygen adaptability verification test of the strains containing the hypoxia-specific expression cassette are as shown in the appendix Figure 11 - 13 The results prove that the Salmonella strains SWT1001, SWT1005, SWT2009, and SWT2013 containing the described hypoxia-specific gene expression cassette exhibit hypoxia regulation ability.

[0423] Example 5: Oxygen concentration simulation of a strain library containing a hypoxia-specific gene expression cassette

[0424] By using an anaerobic gas-generating bag to consume the oxygen in a sealed culture tank and using an oxygen meter to measure the oxygen concentration in the sealed culture tank, after using the anaerobic gas-generating bag for a certain period of time, the oxygen concentration in the sealed culture tank can be fixed within a specific concentration range. The specific method is as follows:

[0425] Put the anaerobic gas-generating bag (brand: Mitsubishi, Japan, product number: D-119) into a 7.0 L sealed culture tank (brand: Mitsubishi, Japan, product number: D-112), and at the same time put an oxygen meter (brand: Meicheng Electrochemistry, product number: OX-100A).

[0426] The above anaerobic gas generation bag combination was used to verify the growth of genetically modified strains under an oxygen concentration of less than 0.8%.

[0427] Since the range of the pathological hypoxia region of tumors is an oxygen concentration lower than 1%, the present invention plans to prove that Salmonella modified with a hypoxia-specific gene expression cassette can still grow normally when the oxygen concentration is lower than 1% (or close to 1%).

[0428] The regulation of the oxygen concentration was completed by an anaerobic gas generation bag. After 1 hour of consumption by the anaerobic gas generation bag, the oxygen concentration measured by an oxygen meter showed an oxygen concentration of 0.8 - 1%. Therefore, in this experiment, the internal hypoxic environment of tumors was simulated to verify the growth of Salmonella on the culture dish after the anaerobic gas generation bag consumed the sealed culture tank for 1 hour, simulating the growth of Salmonella strains containing a hypoxia-specific gene expression cassette in the internal hypoxic environment of tumors.

[0429] The operation process of the spotting experiment is as follows:

[0430] (1) Pick a single clone of the strain containing the hypoxia-specific expression cassette and inoculate it into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0431] (2) After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600 nm.

[0432] (3) Calculate the bacterial liquid volume of 1 OD bacteria according to the following formula, and supplement deionized water to 1 ml.

[0433]

[0434] (4) Take 10 μl of the above bacterial liquid and spot it onto a LB plate containing D-alanine for culture, label it as "1" and repeat it three times, labeled as "a", "b", and "c".

[0435] (5) At the same time, take 10 μl of the above bacterial liquid and spot it onto another LB plate without D-alanine for culture, label it as "1" and spot three replicates, labeled as "a", "b", and "c".

[0436] (6) After dilution in a 10-fold gradient, take 10 μl of the diluted solution and spot it onto the above medium, label it as "2".

[0437] (7) By analogy with this 10-fold gradient dilution until it is labeled to "8".

[0438] (8) Place the two plates in an oxygen environment of 0.8% at 37°C for culture.

[0439] As shown in the appendix Figure 14 As shown, the present invention compares different strains (in the appendixFigure 14 (A) SWT1001, attached Figure 14 (B) STW1005, attached Figure 14 (C) SWT2009, attached Figure 14 (D) The growth of (D) SWT2013) on LB plates without added D-alanine was used to determine the growth status of each strain at oxygen concentrations below 0.8%. Since the medium without added D-alanine, the corresponding Salmonella strains need to rely on their own oxygen regulation system to grow normally. The results confirmed that the strains containing the low-oxygen specific expression cassette that can grow normally in an anaerobic environment can all grow normally at an oxygen concentration below 0.8%.

[0440] Example 6: Construction of Salmonella strains (SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, SWT2013-T7P) with T7 RNA polymerase homologous replacement of the asd gene locus

[0441] (I) Construction of the plasmid pPRO005 for constitutive expression of T7 RNA polymerase controlled by lacUV5

[0442] To construct a constitutively expressed T7 RNA polymerase, the vector pPRO005 was constructed. The process is as attached Figure 15 As shown, the constitutive expression promoter lacUV5 was placed upstream of the RBS sequence (SEQ ID No. 35) of T7 RNA polymerase (SEQ ID No. 4), and a chloramphenicol resistance gene cm (SEQ ID No. 37) with loxp sequences on both sides was added upstream of lacUV5. The purpose is to recombine it into the Salmonella chromosome in the next step for easy screening.

[0443] 1. The plasmid pSWT007 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) was digested with SpeI and NotI, and a fragment of about 1100 bp containing the chloramphenicol resistance gene cm with loxp sequences on both sides was recovered;

[0444] 2. The primers SWTO100 and SWTO101 were directly annealed to generate nicks of NotI and HindIII, and a fragment of about 70 bp including the promoter lacUV5 was recovered;

[0445] 3. The plasmid pPRO004 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) was digested with HindIII and XhoI, and a fragment of about 2690 bp including the T7 RNA polymerase gene was recovered;

[0446] 4. The plasmid pSWT003 (plasmid backbone) was digested with SpeI and XhoI, and a fragment of about 3000 bp was recovered;

[0447] 5. Ligate the above 5 fragments with T4 DNA ligase, transform DH10B competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), coat the LB plate with ampicillin and chloramphenicol double resistance, and screen monoclonal colonies, named pPRO005.

[0448] 6. Extract plasmid pPRO005, take 100 ng and mix it with the restriction enzyme system of NotI and XhoI, incubate at 37 °C for 1 h, and identify it by agarose gel electrophoresis as shown in the appendix. Figure 16 as shown.

[0449] (2) Recombinant lacUV5-controlled constitutive expression cassette of T7 RNA polymerase to the asd gene locus of Salmonella chromosome

[0450] 1. Pick monoclonal colonies of SWT1001, SWT1005, SWT2009, and SWT2013 respectively and inoculate them into 5 ml of LB liquid medium containing 100 μg / ml D-alanine, and place them in a constant temperature shaker at 32 °C and 220 rpm for 16 hours.

[0451] 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.

[0452] 3. Wash the bacterial cells 3 times with sterilized pure water.

[0453] 4. Prepare the PCR products of SWTO102 and SWTO103.

[0454] Mix the primers SWTO102 and SWTO103 with plasmid pPRO005 and the high-fidelity PCR amplification enzyme system and put them into the PCR amplification equipment.

[0455] The amplification program is 95 °C for 2 minutes; 95 °C for 30 seconds, 60 °C for 30 seconds, 72 °C for 60 seconds, cycle 30 times; 72 °C for 10 minutes; 4 °C for 5 minutes.

[0456] Verify the PCR amplification products of SWTO102 and SWTO103 by agarose gel electrophoresis of the amplified PCR products. The verification results are as shown in appendix Figure 17 A.

[0457] Purify and recover the amplified PCR products through the DNA gel recovery system, and measure the DNA concentration and purity of the recovered PCR products by nanodrop.

[0458] 5. Mix the recovered bacterial cells with the PCR products of 100 ng of SWTO102 and SWTO103, and perform electrotransformation at a voltage of 1.8 kV.

[0459] 6. Spread the electrotransformed bacterial cells on a plate containing 25 μg / ml chloramphenicol, 100 μg / ml D-alanine, and 100 μg / ml DAP (diaminopimelic acid), and incubate overnight in a constant temperature incubator at 32 °C until single clone colonies grow.

[0460] 7. Inoculate the monoclonal 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. The chloramphenicol resistance gene is removed by the action of CRE enzyme.

[0461] 8. Identify positive clones by colony PCR, and use SWTO104 and SWTO106, SWTO105 and SWTO107 for identification.

[0462] Verify the PCR amplification products of the target fragments of strains SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P by agarose gel electrophoresis. The verification results are shown in Appendix Figure 17 B.

[0463] Example 7: Construction of eukaryotic drug mRNA / DNA hybrid dual-delivery cytoplasmic green fluorescent protein (EGFP) expression vector pEU010 and control vector pEU011

[0464] 1. Construction of the control vector pEU011 for single delivery of eukaryotic drug mRNA into the cytoplasm

[0465] In this example, to verify the system of single delivery of eukaryotic drug mRNA into the cytoplasm, a control plasmid vector pEU011 carrying eukaryotic expression EGFP as an example, without self-enhancing regulation and without antibiotic resistance selection, was constructed, as shown in Appendix Figure 18 A.

[0466] The pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were used as the balanced lethal control mechanism, and a double phage-derived T7 promoter was used to control the expression of the drug protein (in plasmid pEU011, this element was replaced by EGFP, SEQ ID No. 17) and the membrane-breaking protein gene LLO (SEQ ID No. 13), respectively.

[0467] The specific operation steps are as follows:

[0468] (1) Digest plasmid pPRO006 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with XhoI and NotI, and recover a fragment of about 820 bp, containing the pUC replicon (SEQ ID No. 36);

[0469] (2) Digest plasmid pEU001 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with SpeI and XhoI, and recover a fragment of 2831 bp, containing the Salmonella asd gene and its regulatory region, and the LLO gene and RBS site (SEQ ID No. 15);

[0470] (3) Digest plasmid pEU002 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with NotI and NcoI, and recover a fragment of about 654 bp, containing the first IRES sequence and kozak sequence controlled by the T7 promoter (SEQ ID No. 16);

[0471] (4) Digest plasmid pEGFP-N1 (purchased from BioFeng) with NcoI and XbaI, and recover a fragment of about 731 bp, containing EGFP (SEQ ID No. 17);

[0472] (5) Digest plasmid pEU002 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with XbaI and SpeI, and recover a fragment of about 281 bp, containing the 3'-UTR region, a 50-bp polyA sequence, the T7 terminator and the second T7 promoter (SEQ ID No. 18);

[0473] (6) Ligate the above 5 fragments with T4 DNA ligase, transform the competent cells of SWT1005-T7P, and spread them on a 100 μg / ml D-alanine LB plate to screen for monoclonal colonies, named SWT5015C.

[0474] (7) Pick monoclonal colonies and amplify them with primers SWTO129 and SWTO130; SWTO131 and SWTO132; identify them by agarose gel electrophoresis. As shown in the appendix Figure 19 The generated plasmid is pEU011.

[0475] 2. Construct the eukaryotic drug mRNA / DNA hybrid dual-delivery cytoplasmic expression vector pEU010

[0476] In this example, in order to verify the eukaryotic drug mRNA / DNA hybrid dual-delivery cytoplasmic system, a plasmid vector pEU010 carrying eukaryotic expression of EGFP as an example and without antibiotic resistance selection was constructed, as shown in the appendix Figure 20 as shown.

[0477] The pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were respectively used as the balanced lethal control mechanism, and a T7 promoter derived from double phages was used to respectively control the transcription of the drug protein (in plasmid pEU010, this element was replaced by EGFP, SEQ ID No. 17) and T7 RNA polymerase (SEQ ID No. 20), and the expression of the membrane-breaking protein gene LLO (SEQ ID No. 13).

[0478] The specific operation steps are as follows:

[0479] (1) Digest plasmid pEU011 with XbaI and SacI, and recover a fragment of about 5322 bp;

[0480] (2) Amplify plasmid pEU002 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with primers SWTO133 and SWTO134, recover a fragment of about 613 bp containing the IRES sequence, and recover it with XbaI and EcoRI. This fragment serves as the second IRES sequence (SEQ ID No. 19);

[0481] (3) Amplify plasmid pPRO004 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with primers SWTO135 and SWTO136, recover a fragment of about 2670 bp containing the T7 RNA polymerase sequence, and recover it with EcoRI and SacI. This fragment serves as the T7 RNA polymerase sequence driven by the second IRES sequence (SEQ ID No. 20);

[0482] (4) Ligate the above three fragments with T4 DNA ligase, transform the competent cells of SWT1005-T7P, and spread them on a 100 μg / ml D-alanine LB plate, and screen for monoclonal colonies, named SWT5015;

[0483] (5) Pick monoclonal colonies, amplify them with primers SWTO137 and SWTO134; SWTO137 and SWTO138; and identify them by agarose gel electrophoresis. As shown in the appendix Figure 21 The generated plasmid is pEU010, and the sequence is SEQ ID No. 21.

[0484] 3. Construction of the cytoplasmic expression vector of the control patent US10987432B2

[0485] The structure of the cytoplasmic expression vector of patent US10987432B2 is as shown in the appendix Figure 22As shown, the plasmid pIKDE-EGFP (SEQ ID No. 22) was synthesized according to the sequence provided in the patent and synthesized by Beijing Liuhe BGI Genomics Co., Ltd.

[0486] 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

[0487] 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.

[0488] The specific steps are as follows:

[0489] 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).

[0490] 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 amount and continue incubation.

[0491] 3. After culturing for about 48 hours, the expression intensity of green fluorescence can be observed under a fluorescence microscope, and the proportion of fluorescent cells can be analyzed by flow cytometry.

[0492] The results of the fluorescence microscope are as attached Figure 23 shown, attached Figure 23 A is the BSR-T7 / 5 cell control. Above is under white light, and below is the fluorescence image. The result shows no fluorescence signal; attached Figure 23 B is the BSR-T7 / 5 cells transfected with pEU010. A large number of cells with fluorescence signals were found; attached Figure 23 C is the BSR-T7 / 5 cells transfected with pEU011. Fluorescent signal cells were found, but the number was significantly less than that of Figure 23 B; attached Figure 23 D is the BSR-T7 / 5 cells transfected with pIKDE-EGFP. Fluorescent signal cells were found, but the number was significantly less than that of Figure 23 B.

[0493] The results of flow cytometry analysis are as attached Figure 24 shown. In the FITC channel, it indicates positive for green fluorescent protein, and the P3 gate is set for green fluorescent protein-positive cells. Attached Figure 24 A is the BSR-T7 / 5 cells transfected with pEU010, and the proportion of positive cells is 44.1%; attached Figure 24 B is the BSR-T7 / 5 cells transfected with pEU011, and the proportion of positive cells is 35.7%; attached Figure 24 C is the BSR-T7 / 5 cells transfected with pIKDE-EGFP, and the proportion of positive cells is 34.8%.

[0494] According to the above two experiments, the plasmids of these three systems can all achieve the synthesis of target proteins in background cells with T7 RNA polymerase expression. Compared with the other two control vectors, the eukaryotic drug mRNA / DNA hybrid dual-delivery cytoplasmic expression vector pEU010 has obvious advantages.

[0495] Example 9: Construct the strain SWT5025 carrying the control vector pIKDE-EGFP according to Patent US10987432B2 as a control strain

[0496] 1. Construction of YB1-like Salmonella (SWT005)

[0497] To restore the strain carrying the control vector pIKDE-EGFP in Patent US10987432B2, the first step is to construct the chassis strain of the original YB1.

[0498] The YB1-like Salmonella strain includes aroA gene deficiency and an oxygen regulation system constructed by the forward hypoxia promoter pepT, the essential gene asd, and the reverse hyperoxia promoter sodA.

[0499] (1) Construction of plasmid pSWT004

[0500] ①pSWT004 plasmid as attached Figure 25 As shown, there are chloramphenicol resistance gene cm (SEQ ID No. 39) with loxp sequences on both sides, pepT promoter (SEQ ID No. 41), asd gene (SEQ ID No. 42), and reverse sodA promoter (SEQ ID No. 43).

[0501] 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.

[0502] Strain SWT001 was amplified using primers SWTO13 and SWTO14, and the product was double-cut with XhoI and HindIII and recovered.

[0503] Primers SWTO15 and SWTO16 were directly annealed to generate XhoI and PstI nicks, and then recovered.

[0504] 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).

[0505] ④Extract plasmid pSWT004 and take 100 ng and mix it with SacI and KpnI endonuclease system, incubate at 37℃ for 1 hour, and identify it by agarose gel electrophoresis. Figure 26 shown.

[0506] (2) Construction of recombinant YB1-like Salmonella SWT005 (YB1-like)

[0507] Plasmid pSWT004 was amplified using primers SWTO17 and SWTO18, recovered, and recombined into SWT003 to construct YB1-like Salmonella SWT005.

[0508] ① Inoculate a monoclonal of SWT003 into 5 ml of LB liquid medium and place it in a constant temperature shaker at 32 °C and 220 rpm for 16 hours.

[0509] 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. Then 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.

[0510] ③ Wash the bacterial cells 3 times with sterilized pure water for later use.

[0511] ④ Amplify pSWT004 with primers SWTO17 and SWTO18, purify and recover the PCR product after recovery. Verify the PCR amplification products of the cm - pepT - asd - sodA fragment SWTO17 and SWTO18 by agarose gel electrophoresis. The verification results are as shown in the appendix Figure 27 and measure the concentration and purity of DNA by nanodrop.

[0512] ⑤ Mix the recovered bacterial cells with 100 ng of the PCR product and perform electrotransformation at a voltage of 1.8 kv.

[0513] ⑥ Spread the electrotransformed bacterial cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml DAP (diaminopimelic acid), and incubate overnight in a constant temperature incubator at 32 °C until single - clone colonies grow.

[0514] ⑦ Identify positive clones by colony PCR. Use SWTO93 and SWTO5, SWTO94 and SWTO95 to identify the insertion of the chloramphenicol resistance gene. Verify the results (SWTO93 and SWTO5, SWTO94 and SWTO95) of the PCR amplification products of Salmonella SWT005 by agarose gel electrophoresis as shown in the appendix Figure 28 as shown.

[0515] (3) Oxygen adaptability verification of YB1 - like Salmonella SWT005

[0516] ① Inoculate a monoclonal of YB1 - like Salmonella SWT005 into 5 ml of LB liquid 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.

[0517] ② After culturing, dilute the strain 10 - fold and measure the absorbance (OD value) at 600 nm.

[0518] ③ Calculate the volume of the bacterial suspension (in microliters) of 1 OD bacteria according to the following formula, and supplement deionized water to 1 ml.

[0519]

[0520] ④ Take 10 μl of the above bacterial suspension and spot it onto two LB plates without DAP for culturing, label it as "1", and repeat three times, label them as "a", "b", and "c".

[0521] ⑤ After dilution in a 10-fold gradient, take 10 μl of the diluted solution and spot it onto the above medium, label it as "2".

[0522] ⑥ By analogy with this 10-fold gradient dilution, until it is labeled up to "8".

[0523] ⑦ Place one of the plates in an anaerobic jar at 37 °C. The operation process is to put the anaerobic gas-generating bag (brand: Mitsubishi, Japan, product number: D-119) into a 7.0 L sealed culture jar (brand: Mitsubishi, Japan, product number: D-112), and culture it for 24 hours in an anaerobic environment (oxygen concentration less than 0.01%). Place the other plate in an atmospheric environment (oxygen concentration 21%) at 37 °C and culture it for 24 hours.

[0524] The results of the oxygen adaptability verification test of YB1-like Salmonella SWT005 are as shown in the appendix Figure 29 as follows. From the oxygen adaptability verification data of YB1-like Salmonella SWT005, it can be concluded that YB1-like Salmonella SWT005 can grow normally without oxygen on an LB plate without DAP (as shown in appendix Figure 29 (A)), and there is obvious bacterial strain growth at concentrations 1 (10 - 2 OD), concentration 2 (10 -3 OD), concentration 3 (10 -4 OD), and concentration 4 (10 -5 OD) under aerobic (atmospheric environment) conditions (as shown in appendix Figure 29 (B)).

[0525] Therefore, it can be seen that YB1-like Salmonella SWT005 realizes the regulation of the growth of bacterial strains in an aerobic environment, which is consistent with the previous reports on YB1.

[0526] 2. Construct strain SWT008 of YB1-like Salmonella (SWT005) by introducing an expression system of T7 RNA polymerase controlled by the placUV5 promoter at the chromosomal gmd position on its chassis

[0527] 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.

[0528] The specific steps are as follows:

[0529] (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.

[0530] (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.

[0531] (3) Wash the cells three times with sterilized purified water.

[0532] (4) Preparation of PCR products of SWTO139 and SWTO140.

[0533] Primers SWTO139 and SWTO140 were mixed with plasmid pPRO005 and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0534] 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.

[0535] 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.

[0536] (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.

[0537] (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 incubator until a single clone colony was grown.

[0538] (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.

[0539] (8) Positive clones were identified by colony PCR using SWTO141, SWTO106, SWTO142, and SWTO107.

[0540] The target fragment PCR amplification product of strain SWT008 was verified by agarose gel electrophoresis. The verification results are shown in the attached Figure 30 shown.

[0541] 3. Construction of strain SWT5025 carrying the control vector pIKDE-EGFP

[0542] 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.

[0543] 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.

[0544] The specific steps are as follows:

[0545] (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.

[0546] (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.

[0547] (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, then place it on ice and let it stand for 1 hour.

[0548] (4) Wash the bacterial cells 3 times with sterilized pure water.

[0549] (5) Prepare the PCR amplification products of pPRO013 for SWTO143 and SWTO144.

[0550] Construct the pPRO013 plasmid, as shown in the appendix Figure 31 as follows

[0551] ① Digest the plasmid pPRO009 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with NdeI and PstI, and recover a fragment of about 1600 bp, containing the gene LLO after codon optimization of Salmonella;

[0552] ② Digest the plasmid pPRO011 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with NotI and NdeI, and recover a fragment of about 425 bp, containing the sseA promoter and RBS site sequence, and this fragment is used as the promoter of LLO;

[0553] ③ Digest the plasmid pSWT002 with NotI to recover a 1130 bp fragment;

[0554] ④ Double-digest the plasmid pSWT003 (plasmid backbone) with NotI and PstI, ligate the 4 fragments with T4 DNA ligase, spread them on an LB plate with dual resistance to ampicillin and chloramphenicol, and screen for monoclonal colonies, named pPRO013.

[0555] (6) Mix the primers SWTO143 and SWTO144 with the plasmid pPRO013 and the high-fidelity PCR amplification enzyme system, and place them in a PCR amplification device.

[0556] The amplification program is 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, for 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes.

[0557] Purify and recover the amplified PCR products through a DNA gel recovery system, and measure the concentration and purity of the DNA of the recovered PCR products by nanodrop.

[0558] (7) Mix the recovered bacterial cells with 100 ng of the PCR products of SWTO143 and SWTO144, and perform electrotransformation at a voltage of 1.8 kV.

[0559] (8) Spread the electrotransformed bacteria on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml DAP, and incubate overnight in a constant temperature incubator at 32 °C until single clone colonies grow.

[0560] (9) Inoculate the monoclonal into 5 ml of LB medium containing 100 μg / ml DAP, and culture it in a constant temperature shaker at 37 °C and 220 rpm for 16 hours. Through the action of CRE enzyme, the chloramphenicol resistance gene is removed to generate strain SWT5025.

[0561] (10) Identify positive clones by colony PCR, using SWTO145 and SWTO5, SWTO146 and SWTO116 for identification.

[0562] Verify the PCR amplification product of the target fragment of strain SWT5025 by agarose gel electrophoresis, and the verification results are as shown in the appendix Figure 32 as follows.

[0563] Example 10: Comparison of the performance and cell presentation efficiency of strains SWT5015, SWT5015C and SWT5025

[0564] In this example, the performance of strain SWT5015 of the eukaryotic drug mRNA / DNA mixed double delivery cytoplasmic green fluorescent protein (EGFP) expression vector pEU010 and strain SWT5015C of the control vector pEU011, and strain SWT5025 of pIKDE-EGFP were compared in terms of strain stability, expression rate of membrane-breaking protein and EGFP expression efficiency. The characteristics of these three systems are described in Table 2.

[0565] Table 2 Comparison of the characteristics of strains SWT5015, SWT5015C and SWT5025

[0566]

[0567]

[0568] 1. Comparison of the stability of strains SWT5015, SWT5015C and SWT5025

[0569] Both strains SWT5015 and SWT5025 adopt the self-enhancing regulation mode of eukaryotic-expressed T7 RNA polymerase on the plasmid. Although the self-enhancing eukaryotic-expressed T7 RNA polymerase has a very weak expression ability in host bacteria, due to the constitutively expressed T7 RNA polymerase on the chromosome, the self-enhancing mode is likely to cause cyclic self-enhancing transcription in host bacteria, resulting in the depletion of internal resources and the death of bacteria.

[0570] In this embodiment, by using SWT1005 as a control, the growth and survival status of three strains, SWT5015, SWT5015C, and SWT5025, in a normal culture medium environment was compared to determine the stability of the strains.

[0571] The specific operation steps are as follows:

[0572] (1) Separate monoclonal colonies of SWT1005, SWT5015, and SWT5015C were picked and inoculated into 5 ml of LB liquid medium containing 100 μg / ml D-alanine; monoclonal colonies of SWT5025 were picked and inoculated into 5 ml of LB liquid medium containing 100 μg / ml DAP and 100 μg / ml sodium ampicillin, and then placed in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0573] (2) The cultures of SWT1005, SWT5015, SWT5015C, and SWT5025 were inoculated into fresh liquid medium at a ratio of 1:1000 and transferred to a 96-well cell culture plate, and then continued to be cultured in a constant temperature shaker at 37°C and 220 rpm for another 24 hours. During this period, the bacterial density OD was measured hourly using a microplate reader and recorded, and the growth status is shown in Appendix 600 once and recorded, and the growth status is shown in Appendix Figure 33 A.

[0574] (3) After the cultivation was completed, the strains were diluted 10-fold and the absorbance (OD value) at 600 nm was measured.

[0575] The amount of bacterial liquid per 1 OD was calculated according to the following formula, and deionized water was added to make up to 1 ml.

[0576]

[0577] (4) 10 μl of the above-mentioned SWT1005, SWT5015, and SWT5015C bacterial liquids were spotted onto an LB plate containing D-alanine for cultivation, and 10 μl of the SWT5025 bacterial liquid was spotted onto an LB plate containing DAP for cultivation, marked as "1" and repeated three times, marked as "a", "b", and "c"; after dilution in a 10-fold gradient, 10 μl of the diluted liquid was then spotted onto the above-mentioned medium, marked as "2"; and so on by 10-fold gradient dilution until marked to "8", and the growth status is shown in Appendix Figure 33 B.

[0578] Results showed that there were no differences in the growth rate of SWT5015 and SWT5015C compared with the control strain SWT1005, whether in liquid culture or in plate counting. It was proved that the self-reinforcing regulation mode of SWT5015 had no effect on the strain stability. However, the growth rate of SWT5025 was severely lagged in liquid culture, and flocculent precipitates were found in the culture medium during the culture process, which were dead bacteria in the growth, resulting in the overall OD reading being lower than that of SWT5015, SWT5015C and the control strain SWT1005 in the later stage of culture.

[0579] In addition, during plate counting, it was found that at the same OD, the viable cell count showed that SWT5025 had 100 times fewer viable cells than other strains, only 1% of the viable cell count of other strains. It showed that this self-reinforcing regulation mode of SWT5025 would seriously affect the survival number of bacteria.

[0580] 2. Comparison of EGFP mRNA transcription in SWT5015, SWT5015C and SWT5025 strains

[0581] Since SWT5015, SWT5015C and SWT5025 strains can all present mRNA and constitutively express T7 RNA polymerase, the mRNA of the eukaryotic expression EGFP of the target protein will exist in bacteria. Therefore, in this example, using SWT1005 as a control, the mRNA levels of eukaryotic expression EGFP in these three strains were identified by reverse transcription qPCR.

[0582] The specific operation steps are as follows:

[0583] (1) Respectively pick monoclonal colonies of SWT1005, SWT5015 and SWT5015C and inoculate them into 5 ml of LB liquid medium containing 100 μg / ml D-alanine; pick monoclonal colonies of SWT5025 and inoculate them into 5 ml of LB liquid medium containing 100 μg / ml DAP and 100 μg / ml sodium ampicillin, and place them in a constant temperature shaker at 37 °C and 220 rpm for 16 hours.

[0584] (2) Respectively transfer the cultured strains to centrifuge tubes containing 1 ml of fresh corresponding medium at a ratio of 1:100, and culture them at 32 °C and 200 rpm for 4 - 6 hours until OD 600 is between 0.5 and 1.

[0585] (3) Centrifuge the bacterial liquid of each strain at 4 °C and 12,000 rpm for 2 minutes, and carefully remove all the supernatant.

[0586] (4) Resuspend the cells thoroughly with 100 μl of TE buffer containing lysozyme (purchased from Shanghai Beyotime Biotechnology Co., Ltd., product number ST206) by pipetting up and down, and incubate at room temperature for 5 minutes.

[0587] (5) Add 300 μl of Lysis buffer (GeneJET RNA Purification Kit, purchased from Thermo, product number K0731) to each tube, and vortex to mix well.

[0588] (6) Add 180 μl of absolute ethanol to each tube, and pipette to mix well. Transfer all the liquid in the centrifuge tube to an RNA purification column (GeneJET RNA Purification Kit, purchased from Thermo, product number 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.

[0589] (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.

[0590] (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.

[0591] (9) Transfer the adsorption column to a new 1.5 ml centrifuge tube, add 50 μl of DEPC water (purchased from Shanghai Beyotime 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.

[0592] (10) Place the RNA tube on ice, measure its concentration and purity by Nanodrop, and then take 500 ng for 1% electrophoresis to detect its integrity. The identification results are shown in Appendix Figure 34 A.

[0593] (11) Remove the DNA residue in the RNA. Pipette 1 μl of the extracted RNA, add 1 μl of DNase I (purchased from Thermo, product 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.

[0594] (12) Pipette 1 μl of the RNA from the previous step, mix it into the corresponding primer SWTO147 and SWTO148, and the Luna Universal qPCR Master Mix (purchased from NEB, product number M3003S) amplification enzyme system, and then mix and place it in a fluorescence PCR amplification device.

[0595] The amplification program was 95°C for 60 seconds; 95°C for 15 seconds, 60°C for 30 seconds, with 40 cycles.

[0596] (13) The reverse transcription process was not performed in this step. The purpose was to exclude the interference of residual DNA in the experiment. The results are shown in Appendix Figure 34 B.

[0597] (14) Pipette 1 μl of the RNA from step (11) and mix it into the amplification enzyme system of the corresponding primers SWTO147 and SWTO148 Luna Universal One-step RT-qPCR kit (purchased from NEB, catalog number E3005S), and then mix and place it in a fluorescence PCR amplification device.

[0598] The amplification program was 55°C for 10 minutes; 95°C for 60 seconds; 95°C for 15 seconds, 60°C for 30 seconds, with 40 cycles. The purpose was to first convert mRNA into cDNA through reverse transcription and then amplify the target gene. The results are shown in Appendix Figure 34 B.

[0599] The above experimental results showed that the mRNAs of the target gene EGFP were all contained in strains SWT5015, SWT5015C, and SWT5025, and there were no significant differences in the levels of their mRNAs.

[0600] 3. Comparison of the expression efficiencies of T7 RNA polymerase and lysis protein LLO in strains SWT5015, SWT5015C, and SWT5025

[0601] The T7 RNA polymerases 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 in 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 storing a sufficient amount of LLO protein in bacteria can maximize the delivery efficiency of the overall bacteria. While in SWT5025, it is controlled by the promoter (SEQ ID No. 40) of the sseA gene on Salmonella pathogenicity island II on the chromosome and is induced to express after the bacteria invade the cells.

[0602] In this example, verify the expression of T7 RNA polymerase and LLO protein in strains SWT5015, SWT5015C, and SWT5025 in a normal medium and in the sseA promoter-induced medium N-salts (containing low concentrations of magnesium ions that can mimic the environment after cell invasion and induce the expression of the sseA promoter; the composition is 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 100 mM Bis-Tris / HCl (pH 7.0), 38 mM glycerol, and 0.1% Casamino Acids) with the addition of 30 μM MgCl2).

[0603] The specific operating steps are as follows:

[0604] (1) Inoculate single colonies of SWT5015 and SWT5015C into 5 ml of LB liquid medium containing 100 μg / ml D-alanine; inoculate a single colony of SWT5025 into 5 ml of LB liquid medium containing 100 μg / ml DAP and 100 μg / ml sodium ampicillin, and place them in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0605] (2) Take 1 ml of the bacterial liquid of each strain, centrifuge at 12,000 RPM for 1 minute, discard the supernatant, recover the precipitate, and set aside.

[0606] (3) Take 1 ml of the bacterial liquid of each strain, centrifuge at 4,000 RPM for 5 minutes, discard the supernatant, resuspend in 5 ml of freshly prepared induction medium N-salts. Add 100 μg / ml D-alanine to the media of SWT5015 and SWT5015C; add 100 μg / ml DAP and 100 μg / ml sodium ampicillin to the medium of SWT5025. Then place them in a constant temperature shaker at 37°C and 220 rpm for 24 hours.

[0607] (4) Take 5 ml of the bacterial liquid of each strain after culturing in the induction medium N-salts, centrifuge at 12,000 RPM for 1 minute, discard the supernatant, recover the precipitate, and set aside.

[0608] (5) Resuspend the samples in steps (2) and (4) using a lysis buffer (protein loading buffer, purchased from CST, catalog number 7722S), heat at 95°C for 10 minutes, centrifuge again at 12,000 rpm, and recover the supernatant for use.

[0609] (6) Pipette 5 μl of the supernatant recovered in the previous step and add it to an SDS-PAGE gel (the SDS-PAGE gel preparation kit is purchased from Sangon Biotech, product number C631100-0200), and perform electrophoresis for 60 minutes under the condition of 120 V.

[0610] (7) Transfer the SDS-PAGE gel after electrophoresis to a PVDF membrane (purchased from Roche, product number 03010040001) under the condition of a constant current of 250 mA for 30 minutes.

[0611] (8) Transfer the PVDF membrane after transfer to 5% skim milk powder, place it on a horizontal shaker, incubate at 80 rpm at room temperature for 1 hour as a blocking method.

[0612] (9) Use Anti-T7 RNAPolymerase antibody (T7 RNA polymerase antibody, purchased from Creative Biomart, product number CABT-B8990) and Anti-listeriolysin antibody (identification antibody for LLO, purchased from abcam, product number ab200538) as primary antibodies and dilute them with 5% skim milk powder and incubate at 4 °C for 18 hours (each membrane uses a single antibody alone).

[0613] (10) Dilute the secondary antibody with 5% skim milk powder, transfer the PVDF membrane after incubation with the primary antibody to the secondary antibody dilution solution, place it on a horizontal shaker, incubate at 80 rpm at room temperature for 1 hour.

[0614] (11) Wash the PVDF membrane after incubation with the secondary antibody 3 times with TBST Buffer (purchased from Sangon Biotech, product number C520009-0005), 5 minutes each time. Drop ECL developer (developer SignalFire TM ECLReagent is purchased from CST, product number 6883p3) onto the washed PVDF membrane and incubate in the dark for 1 min. After incubation, perform image acquisition of the PVDF membrane with a chemiluminescence imager.

[0615] Under the culture conditions of LB and N-salts, the strains SWT5015, SWT5015C, and SWT5025 identified by Western Blot can all highly express T7 RNA polymerase, as shown in Figure 35 Figure A; under the culture conditions of LB and N-salts, the strains SWT5015 and SWT5015C identified by Western Blot can all highly express LLO, as shown in Figure 35 Figure B; SWT5025 can only express LLO under the culture condition of N-salts, as shown in Figure 35As shown in Figure B. The spatio-temporal expression difference of LLO may affect the efficiency of presenting mRNA / DNA after bacterial invasion.

[0616] 4. Comparison of the expression efficiency of EGFP after the invasion of cancer cells by strains SWT5015, SWT5015C and SWT5025

[0617] In the above embodiments, the stability of SWT5015, SWT5015C and SWT5025, as well as the expression efficiency of T7 polymerase and LLO were verified respectively. Although differences have been found, cell invasion experiments need to be used to further judge the drug delivery efficiency.

[0618] In this embodiment, SWT1005 was used as a negative control, and strains SWT5015, SWT5015C and SWT5025 were co-cultured with CT26 cancer cells respectively, and the differences in efficiency were analyzed from the perspective of the expression of fluorescent protein.

[0619] The specific operation steps are as follows:

[0620] (1) Single colonies of SWT1005, SWT5015 and SWT5015C were picked and inoculated into 5 ml of LB liquid medium containing 100 μg / ml D-alanine; single colonies of SWT5025 were picked and inoculated into 5 ml of LB liquid medium containing 100 μg / ml DAP and 100 μg / ml sodium ampicillin, and placed in a constant temperature shaker at 37 °C and 220 rpm for 16 hours.

[0621] (2) Measure 1 OD of the culture and wash it 3 times with PBS.

[0622] (3) Add 2.5×10 6 CT26 cancer cells into each well of a 6-well plate for cell culture, and place them in a 5% CO2 incubator at 37 °C overnight in complete medium (DMEM basal medium + 10% fetal bovine serum + double antibodies).

[0623] (4) After counting the cells in 2 wells, add strains SWT1005, SWT5015, SWT5015C and SWT5025 respectively according to the cell number, with an infection ratio of 200:1. Place them in an anaerobic jar with an oxygen concentration below 0.8% and co-culture for 2 hours to allow the bacteria to invade the cells. Then carefully wash the cells with an equal volume of PBS, aspirate the supernatant, add DMEM medium containing 100 μg / mL gentamicin, and continue to culture in an anaerobic jar with an oxygen concentration below 0.8% for 24 hours. Then take pictures under a fluorescence microscope as shown in Appendix Figure 36 A.

[0624] (5) Digest the invaded cells with 0.25% trypsin (purchased from Shanghai Yuanpei Biotech Co., Ltd., product number S310JV). After resuspending with 100 μl of PBS, transfer them to a black flat-bottom 96-well plate and place them in a microplate reader to read the green fluorescence signal value at room temperature (emission wavelength 518 nm, absorption wavelength 518 nm, fixed reading time 800 ms). The results are shown in Figure 36 Figure B as shown below.

[0625] As can be seen from the above results, through the analysis of the microplate reader, SWT1005 as a negative control has no fluorescence signal; the strains SWT5015, SWT5015C, and SWT5025 can all detect the signal of EGFP, and among them, the signal of SWT5015 is the strongest. Due to the low efficiency of the eukaryotic system presented by bacteria, only the efficiency of the mRNA / DNA mixed dual delivery of cytoplasm by SWT5015 is high enough to clearly observe the fluorescence signal under a fluorescence microscope. Therefore, it can be judged that the delivery efficiency of SWT5015 is the best.

[0626] Example 11: Construction of eukaryotic drug mRNA / DNA mixed dual delivery of cytoplasm vectors pEU013 and presenting strains SWT5115 and SWT5119 with GSDMD-N, construction of vectors pEU014 and presenting strains SWT5215 and SWT5219 with GSDME-N, and verification of mRNA transcription of the above strains

[0627] In this example, in order to verify the ability of eukaryotic drug mRNA / DNA mixed dual delivery of cytoplasm to deliver GSDM-N, a vector pEU013 with GSDMD-N and presenting strains SWT5115 and SWT5119, as well as a vector pEU014 with GSDME-N and presenting strains SWT5215 and SWT5219 were constructed.

[0628] 1. Construction of plasmid vector pEU013 and strains SWT5115 and SWT5119

[0629] The pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were respectively used as the balanced lethal control mechanism, and a T7 promoter derived from a double phage was used to respectively 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).

[0630] The specific operation steps are as follows:

[0631] (1) Digest the plasmid pEU010 with NcoI and XbaI, and recover a fragment of about 7869 bp;

[0632] (2) Digest the plasmid pEU003 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with NcoI and XbaI, and recover a fragment of about 828 bp, containing the GSDMD-N sequence;

[0633] (3) Ligate the above two fragments with T4 DNA ligase, transform the competent cells of SWT1005-T7P, and spread them on a 100 μg / ml D-alanine LB plate, and screen for monoclonal colonies, named SWT5115.

[0634] (4) Pick monoclonal colonies and amplify them with primers SWTO129 and SWTO149; SWTO150 and SWTO151; identify them by agarose gel electrophoresis, as shown in the appendix Figure 37 The generated plasmid is pEU013.

[0635] (5) Extract the plasmid pEU013, transform the competent cells of SWT2009-T7P, and spread them on a 100 μg / ml D-alanine LB plate, and screen for monoclonal colonies, named SWT5119.

[0636] 2. Construction of plasmid vector pEU014 and strains SWT5215 and SWT5219

[0637] Respectively use the pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) as a balanced lethal control mechanism, and use a T7 promoter derived from a double phage to control the transcription of the drug protein GSDME-N (SEQ ID No. 24) and the expression of the T7 RNA polymerase (SEQ ID No. 20) and the membrane-breaking protein gene LLO (SEQ ID No. 13).

[0638] The specific operation steps are as follows:

[0639] (1) Digest the plasmid pEU010 with NcoI and XbaI, and recover a fragment of about 7869 bp;

[0640] (2) Digest the plasmid pEU004 (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.) with NcoI and XbaI, and recover a fragment of about 816 bp, containing the GSDME-N sequence;

[0641] (3) Ligate the above two fragments with T4 DNA ligase, transform the competent cells of SWT1005-T7P, spread them on the 100 μg / ml D-alanine LB plate, and screen for monoclonal colonies, named SWT5215;

[0642] (4) Pick monoclonal colonies and amplify them with primers SWTO129 and SWTO152; SWTO150 and SWTO153; identify them by agarose gel electrophoresis. As shown in the appendix Figure 37 The generated plasmid is pEU014.

[0643] (5) Extract plasmid pEU014, transform the competent cells of SWT2009-T7P, spread them on the 100 μg / ml D-alanine LB plate, and screen for monoclonal colonies, named SWT5219.

[0644] 3. Verify the mRNA transcription of GSDMD-N in strains SWT5115 and SWT5119, and GSDME-N in strains SWT5215 and SWT5219

[0645] Since strains SWT5115, SWT5119, SWT5215, and SWT5219 can all present mRNA and constitutively express T7 RNA polymerase, the mRNA of the eukaryotic expression of the target proteins GSDMD-N and GSDME-N will exist in the bacteria. Therefore, in this example, SWT1005 was used as a control, and the mRNA levels of eukaryotic-expressed GSDMD-N and GSDME-N in strains SWT5115 and SWT5215 were identified by reverse transcription qPCR; meanwhile, in this example, SWT2009 was used as a control, and the mRNA levels of eukaryotic-expressed GSDMD-N and GSDME-N in strains SWT5119 and SWT5219 were identified by reverse transcription qPCR.

[0646] The specific operation steps are as follows:

[0647] (1) Pick monoclonal colonies of SWT1005, SWT5115, SWT5215; SWT2009, SWT5119, and SWT5219 respectively and inoculate them into 5 ml of LB liquid medium containing 100 μg / ml D-alanine, and place them in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0648] (2) Transfer the cultured strains to centrifuge tubes containing 1 ml of fresh corresponding medium at a ratio of 1:100, and culture them at 32°C and 200 rpm for 4-6 hours until the OD 600 is between 0.5 and 1.

[0649] (3) Centrifuge the bacterial suspensions of each strain at 4°C and 12,000 rpm for 2 minutes, and carefully remove all the supernatants.

[0650] (4) Resuspend the cells thoroughly with 100 μl of TE buffer containing lysozyme (purchased from Beyotime Biotechnology Co., Ltd., Shanghai, catalog number ST206) by pipetting up and down, and incubate at room temperature for 5 minutes.

[0651] (5) Add 300 μl of Lysis buffer (GeneJET RNA Purification Kit, purchased from Thermo, catalog number K0731) to each tube and mix well by vortexing.

[0652] (6) Add 180 μl of absolute ethanol to each tube and mix well by pipetting. Transfer all the liquid in the centrifuge tube to an RNA purification column (GeneJET RNA Purification Kit, purchased from Thermo, catalog number 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.

[0653] (7) Add 700 μl of washbuffer1 (GeneJET RNA Purification Kit, purchased from Thermo, catalog number K0731) to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0654] (8) Add 600 μl of washbuffer2 (GeneJET RNA Purification Kit, purchased from Thermo, catalog number K0731) to the adsorption column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0655] (9) Transfer the adsorption column to a new 1.5-ml centrifuge tube, add 50 μl of DEPC water (purchased from Beyotime Biotechnology Co., Ltd., Shanghai, catalog number R0022), let it stand at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, and discard the adsorption column.

[0656] (10) Place the RNA tube on ice. After detecting its concentration and purity with Nanodrop, take 500 ng and run a 1% electrophoresis to detect its integrity. The identification results are as shown in Figure 34 Figure A.

[0657] (11) Remove the DNA residue in the RNA. Pipette 1 μl of the 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.

[0658] (12) Pipette 1 μl of the RNA from the previous step and mix it into the corresponding primers SWTO154 and SWTO155 (for identifying the mRNA of GSDMD-N) and SWTO156 and SWTO157 (for identifying the mRNA of GSDME-N) and the Luna Universal qPCR MasterMix (purchased from NEB, catalog number M3003S) amplification enzyme system, and then mix and place it into a fluorescence PCR amplification device.

[0659] The amplification program is 60 seconds at 95 °C; 15 seconds at 95 °C, 30 seconds at 60 °C, for 40 cycles;

[0660] (13) The reverse transcription process was not performed in this step. The purpose was to exclude the interference of residual DNA in the experiment. The results are as attached Figure 38 as shown.

[0661] (14) Pipette 1 μl of the RNA from step (11) and mix it into the corresponding primers SWTO154 and SWTO155 (for identifying the mRNA of GSDMD-N) and SWTO156 and SWTO157 (for identifying the mRNA of GSDME-N) and the Luna Universal One-step RT-qPCR kit (purchased from NEB, catalog number E3005S) amplification enzyme system, and then mix and place it into a fluorescence PCR amplification device.

[0662] The amplification program is 10 minutes at 55 °C; 60 seconds at 95 °C; 15 seconds at 95 °C, 30 seconds at 60 °C, for 40 cycles. The purpose is to first convert mRNA into cDNA through reverse transcription and then amplify the target gene. The results are as attached Figure 38 as shown.

[0663] The above experimental results show that the strains SWT5115 and SWT5215 respectively contain the mRNA transcribed from the first expression unit of the target genes GSDMD-N (attached Figure 38 A) and GSDME-N (attached Figure 38 B), and there is no significant difference in the level of their mRNA. The strains SWT5119 and SWT5219 respectively contain the mRNA transcribed from the first expression unit of the target genes GSDMD-N (attached ​ C) and GSDME-N (attached ​ D), and there is no significant difference in the level of their mRNA.

[0664] The results of this example show that using strains with different hypoxia-specific expression cassettes has no effect on the mRNA transcribed from their first expression units.

[0665] Example 12: Verification of the pyroptosis and killing effects of strains SWT5115 and SWT5215 on various types of cancer cells

[0666] Co-cultured with cancer cells under in vitro hypoxic conditions to verify the killing effects of strains SWT5115 and SWT5215 on various cancer cells, and strain SWT1005 was used as a control strain.

[0667] The specific steps are as follows:

[0668] 1. Streak strains SWT1005, SWT5115, and SWT5215 on LB plates containing 100 μg / ml D-alanine and statically culture overnight in a 37 °C incubator.

[0669] 2. Pick monoclonal colonies of strains SWT1005, SWT5115, and SWT5215 and inoculate them into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and place them in a constant temperature shaker at 37 °C and 220 rpm for 16 hours.

[0670] 3. Measure the absorbance of the strains 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 medium, and resuspend the bacteria in the cell medium (infection ratio is 200:1).

[0671] 4. Co-culture the above bacteria 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), osteosarcoma (K7M2) under an oxygen concentration of less than 0.8% for 2 hours, then wash the cells 3 times with PBS containing gentamicin, change the medium, and continue to culture in an anaerobic jar under an oxygen concentration of less than 0.8% for 24 hours, and take pictures and record them with a 40× objective lens under a white light field of view, as shown in the appendix ​ as follows.

[0672] 5. Mix the CCK8 reagent with the above cell suspension and incubate for 1 hour.

[0673] 6. Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (detection wavelength 450 nm) and calculate the percentage of cytotoxicity.

[0674] 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 the appendix ​ as follows.

[0675] Among them, the experimental results were analyzed by T-test, and p < 0.05 (*), p < 0.01 (**), p <= 0.001 (***) indicated significant differences.

[0676] The above results show that the Salmonella strains SWT5115 and SWT5215 carrying the expression vector have obvious killing effects on various cancer cells. Significant cell pyroptosis can be found under a microscope under white light (indicated by arrows), while SWT1005 shows no obvious pyroptosis.

[0677] In addition, it was found through the analysis of the death reagent CCK8 that their killing effect on cells is stronger than that of the control strain SWT1005, indicating that Salmonella with eukaryotic drug mRNA / DNA mixed double-delivery cytoplasmic delivery of GSDM-N has universality in cell pyroptosis and killing of cancer cells.

[0678] Example 13: Verification of the pyroptosis and killing effects of strains SWT5119 and SWT5219 on various types of cancer cells

[0679] Co-cultured with cancer cells under in vitro hypoxic conditions to verify the killing effects of strains SWT5119 and SWT5219 on various cancer cells, with strain SWT2009 as the control strain.

[0680] The specific steps are as follows:

[0681] 1. Streak strains SWT2009, SWT5119, and SWT5219 on an LB plate containing 100 μg / ml D-alanine and statically culture overnight in a 37 °C constant temperature incubator.

[0682] 2. Pick single colonies of strains SWT2009, SWT5119, and SWT5219 and inoculate them into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and place them in a constant temperature shaker at 37 °C and 220 rpm for 16 hours.

[0683] 3. Measure the absorbance of the strains at 600 nm, take the culture with an OD value of 1, wash it twice with PBS, and then wash it once with the corresponding cell medium, and resuspend the cell pellet in the cell 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 the cells were cultured in an anaerobic jar at an oxygen concentration below 0.8% for 24 hours. The images were taken with a 40x objective lens under white light, as shown in the attached figure. ​ shown.

[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 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 the attached table. ​ shown.

[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 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.

[0690] 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.

[0691] 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.

[0692] Example 14: Evaluation of the tumor inhibitory effects of strains SWT5115, SWT5215 and control strain SWT1005 in a mouse tumor model

[0693] To verify the inhibitory effect of the strain carrying the expression vector on the growth of mouse tumors, the present invention designed inhibitory tests of SWT5115 and SWT5215 as representatives in mouse EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7 and MB49 tumor models.

[0694] 1. Establishment of various mouse tumor models

[0695] (1) Establishment of the EMT6 tumor mouse model

[0696] BALB / c mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a Specific Pathogen Free (SPF) environment) were subcutaneously inoculated with 1×10 6 EMT6 cancer cells to establish a subcutaneous mammary tumor model in mice. 14-18 days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 125 μl of 1×10 7 CFU bacteria.

[0697] (1) Establishment of the K7M2 tumor mouse model

[0698] BALB / c mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a Specific Pathogen Free (SPF) environment) were subcutaneously inoculated with 1×10 6 K7M2 cancer cells to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 125 μl of 1×10 7 CFU bacteria.

[0699] (2) Establishment of the Hepa1-6 tumor mouse model

[0700] C57BL / 6 mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Hepa1-6 cancer cells to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 125 μl of 1×10 7 CFU bacteria.

[0701] (3) Establishment of A549 tumor mouse model

[0702] Nu / Nu nude mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 A549 cancer cells to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 125 μl of 1×10 7 CFU bacteria.

[0703] (4) Establishment of B16F10 tumor mouse model

[0704] C57BL / 6 mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 B16F10 cancer cells to establish a subcutaneous mammary tumor model in mice. 14-18 days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 125 μl of 1×10 7 CFU bacteria.

[0705] (5) Establishment of Renca tumor mouse model

[0706] BALB / c mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Renca cancer cells to establish a subcutaneous tumor model in mice. 14 - 18 days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0707] (6) Establishment of MFC tumor mouse model

[0708] BALB / c mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 MFC cancer cells to establish a subcutaneous tumor model in mice. 14 - 18 days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0709] (7) Establishment of Pan02 tumor mouse model

[0710] C57BL / 6 mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Pan02 cancer cells to establish a subcutaneous tumor model in mice. 14 - 18 days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0711] (8) Establishment of RM-1 tumor mouse model

[0712] C57BL / 6 mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 RM-1 cancer cells to establish a subcutaneous tumor model in mice. Fourteen to eighteen days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into a PBS control group, SWT1005, SWT5115, and SWT5215, a total of 4 groups, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0713] (9) Establishment of CT26 tumor mouse model

[0714] BALB / c mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 CT26 cancer cells to establish a subcutaneous tumor model in mice. Fourteen to eighteen days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into a PBS control group, SWT1005, SWT5115, and SWT5215, a total of 4 groups, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0715] (10) Establishment of ID8 tumor mouse model

[0716] C57BL / 6 mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 ID8 cancer cells to establish a subcutaneous tumor model in mice. Fourteen to eighteen days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into a PBS control group, SWT1005, SWT5115, and SWT5215, a total of 4 groups, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0717] (11) Establishment of Neuro-2a tumor mouse model

[0718] A / J mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Neuro-2a cancer cells to establish a subcutaneous tumor model in mice. Fourteen to eighteen days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0719] (12) Establishment of SCC7 tumor mouse model

[0720] Nu / Nu nude mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 SCC7 cancer cells to establish a subcutaneous tumor model in mice. Fourteen to eighteen days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0721] (13) Establishment of MB49 tumor mouse model

[0722] C57BL / 6 mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 MB49 cancer cells to establish a subcutaneous tumor model in mice. Fourteen to eighteen days after inoculation, when the tumor volume was about 100 mm 3 , the experiment was carried out. The mice were divided into 4 groups: PBS control group, SWT1005, SWT5115, and SWT5215, with 5 mice in each group. The tumor-bearing mice were inoculated via the tail vein with 1×10 7 CFU bacteria in a volume of 125 μl.

[0723] 2. Preparation and treatment of bacteria

[0724] Pick the constructed Salmonella strains SWT1005, SWT5115, and SWT5215 monoclonal and inoculate them into 5 ml of LB liquid medium supplemented with D-alanine, and place them in a constant temperature shaker at 37 °C and 220 rpm for 16 hours; measure the absorbance of the strains at 600 nm, take the culture with an OD value of 1, and wash it 3 times with PBS; inject 125 μl of 1×10 7 CFU of bacteria-administered model mice; stipulate that the day of administration is day 0, and measure the length and width of the mouse tumors on days 1, 3, 5, 7, 9, and 11 after administration, and calculate the mouse tumor volume according to the following formula.

[0725] Mouse tumor volume = length × width 2 × 0.52

[0726] Compared with SWT1005, SWT5115, and SWT5215, the results of tumor inhibition in the mouse tumor model are as shown in the appendix ​ as follows.

[0727] Among them, the experimental results were analyzed by t-test, p < 0.05 (*), p < 0.01 (**), p <= 0.001 (***) indicating significant differences.

[0728] The results showed that the Salmonella strains SWT5115 and SWT5215 carrying the expression vector had an obvious effect of inhibiting tumor growth, which was better than the control strain SWT1005 and the untreated control.

[0729] The information of the corresponding strains of the present invention is shown in Table 3

[0730] Table 3 Strain construction table

[0731]

[0732]

[0733] The tool plasmid of the present invention is shown in Table 4.

[0734] Table 4 Tool plasmid used in the present invention

[0735]

[0736]

[0737]

[0738] The sequences of the promoter, gene coding region, protein, etc. used in the present invention are shown in Table 5.

[0739] Table 5 Specific sequences of the promoter, gene coding region, protein, etc. used in the present invention

[0740]

[0741]

[0742]

[0743]

[0744]

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756]

[0757] The specific sequences of all the primers used in the present invention are shown in Table 6.

[0758] Table 6 Specific sequences of all the primers used in the present invention

[0759]

[0760]

[0761]

[0762] The above has described the present application in combination with preferred embodiments. However, these embodiments are only exemplary and only serve an illustrative purpose. On this basis, various substitutions and improvements can be made to the present application, and these all fall within the protection scope of the present application.

Claims

1. A eukaryotic expression vector, which comprises a first expression unit and a second expression unit, wherein the first expression unit comprises a first promoter derived from a phage, a eukaryotic drug protein gene expression module, and a phage-derived RNA polymerase gene transcription module, and the second expression unit comprises a second promoter derived from a phage and a transmembrane protein gene expression module. The first promoter controls the expression of the eukaryotic drug protein gene and the transcription of the phage-derived RNA polymerase gene respectively, and the second promoter controls the expression of the transmembrane protein gene. And the phage-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 a phage are selected from T7, T3, and SP6 promoters.

3. The eukaryotic expression vector according to claim 2, wherein the first promoter and the second promoter derived from a phage are T7 promoters.

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

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

6. The eukaryotic expression vector according to any one of the above claims, which comprises the essential gene asd of Salmonella, and the asd gene on the host bacterial chromosome is deleted.

7. The eukaryotic expression vector according to any one of the above claims, which comprises a replicon essential for plasmid replication.

8. The eukaryotic expression vector according to claim 7, wherein the replicon is selected from pUC, p15A, ColE1, and R6K.

9. The eukaryotic expression vector according to claim 8, wherein the replicon is pUC.

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

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

12. The eukaryotic expression vector according to claim 11, wherein the drug protein gene is the N-terminal 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 above claims, wherein the membrane-breaking protein gene is selected from the Listeria hemolysin hlyA gene.

15. The eukaryotic expression vector according to any one of the above claims, wherein the mode of presenting the eukaryotic expression vector is the cytoplasmic expression mode of mRNA / DNA mixture, that is, the eukaryotic expression vector DNA and the mRNA transcribed from the first expression unit are mixed and presented into eukaryotic cells by the host bacterium, and cytoplasmic expression is achieved.

16. The eukaryotic expression vector according to claim 15, which adopts a "self-enhancing" 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 to form a cycle.

17. The eukaryotic expression vector according to any one of the above 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) Connecting in sequence the first promoter derived from a phage, the eukaryotic drug protein gene expression module, and the phage-derived RNA polymerase gene transcription module according to any one of the above claims to construct a first expression unit; (2) Connecting in sequence the second promoter derived from a phage and the membrane-breaking protein gene expression module according to any one of the above claims to construct a second expression unit; (3) Connecting the plasmid replicon, the asd gene, and the above two expression units 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 the first promoter and the second promoter derived from a phage.

21. The Gram-negative bacterium according to claim 20, wherein the specific phage RNA polymerase gene is selected from the T7 RNA polymerase gene, the T3 RNA polymerase gene, and the SP6 RNA polymerase gene.

22. The Gram-negative bacterium according to claim 21, wherein the specific phage RNA polymerase gene is the T7 RNA polymerase gene.

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

24. The Gram-negative bacterium according to claim 23, wherein the constitutive expression promoter is the lacUV5 promoter.

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

26. The Gram-negative bacterium according to claim 25, wherein: The FNR binding site of the forward hypoxic promoter or the reverse hyperoxic 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 bases replaced does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; The ArcA binding site of the reverse hyperoxic promoter conforms to the pattern of GTTAATTA, and any base therein can be replaced, but the total number of bases replaced does not exceed 2.

27. The Gram-negative bacterium according to claim 25 or 26, wherein the forward hypoxic promoter and / or the reverse hyperoxic promoter of the hypoxic-specific gene expression cassette is a promoter derived from a Gram-negative bacterium.

28. The Gram-negative bacterium according to any one of claims 25-27, wherein The forward hypoxic promoter is selected from the promoter region sequences of yhbU or ynfK; The essential gene for survival is selected from alr or dadX; The reverse hyperoxic promoter is selected from the promoter region sequences of cyoA or ydcI.

29. The Gram-negative bacterium according to claim 28, wherein the forward hypoxic promoter is yhbU, which is selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, and Simiduia.

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

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

32. The Gram-negative bacterium according to claim 28, wherein the hypoxic-specific gene expression cassette is composed of the forward hypoxic promoter yhbU, the essential gene for survival alr, and the reverse hyperoxic promoter cyoA.

33. The Gram-negative bacterium according to claim 28, wherein the hypoxic-specific gene expression cassette is composed of the forward hypoxic promoter yhbU, the essential gene for survival alr, and the reverse hyperoxic promoter ydcI.

34. The Gram-negative bacterium according to claim 28, wherein the hypoxic-specific gene expression cassette is composed of the forward hypoxic promoter ynfK, the essential gene for survival dadX, and the reverse hyperoxic promoter cyoA.

35. The Gram-negative bacterium according to claim 28, wherein the hypoxic-specific gene expression cassette is composed of the forward hypoxic promoter ynfK, the essential gene for survival dadX, and the reverse hyperoxic promoter ydcI.

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

37. The Gram-negative bacterium according to claim 36, wherein the forward hypoxia promoter functions when the oxygen content is below 1%, and does not function when the oxygen content is above 1%; and / or the reverse hyperoxia promoter functions when the oxygen content is above 1%, and does not function when the oxygen content is below 1%.

38. The Gram-negative bacterium according to claim 37, wherein the forward hypoxia promoter functions when the oxygen content is below 0.8%, and does not function when the oxygen content is above 0.8%; and / or the reverse hyperoxia promoter functions when the oxygen content is above 0.8%, and does not function when the oxygen content is below 0.8%.

39. A method for controlling the expression of a pharmaceutical protein in a prokaryotic cell using a eukaryotic expression vector according to any one of claims 1-17, 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 the host bacterium.

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

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

42. The Gram-negative bacterium according to any one of the above claims, which is selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simiduia, Enterobacter ludwigii, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Bordetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, Vibrio parahaemolyticus, Vibrio cholerae, Yersinia pestis, Moraxella catarrhalis, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolytica, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori.

43. 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 according to claim 44, wherein the attenuated Gram-negative bacterium is Salmonella.

46. The Gram-negative bacterium according to any one of the above claims, wherein the eukaryotic expression vector is presented in a cytoplasmic expression mode of mRNA / DNA mixture, that is, the eukaryotic expression vector DNA and the mRNA transcribed from the first expression unit are presented into eukaryotic cells by the host bacterium and cytoplasmic expression is achieved.

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

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

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

50. The pharmaceutical composition according to any one of claims 47-49, which is used for treating solid tumors.

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

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

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

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, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

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