Hypoxia-specific gene expression cassette, bacteria containing it, and use

Abstract

This invention provides a hypoxia-specific gene expression cassette, Gram-negative bacteria containing the expression cassette, and their use in the preparation of antitumor drugs and in tumor treatment. This invention rationally designs a forward hypoxia promoter and a reverse hyperxia promoter, and combines them with appropriate survival-essential genes to obtain a more efficient and safer hypoxia-specific gene expression cassette. Gram-negative bacteria modified with the hypoxia-specific gene expression cassette designed in this invention exhibit a lower overall mutation rate. In a mouse tumor model, the bacteria are more widely distributed within the tumor and are eliminated more quickly from normal organs.

Description

[Technical Field]

[0001] This application claims priority to a prior application filed with the China National Intellectual Property Administration on September 19, 2023, with patent application number 2023112106010, titled "Hypoxia-Specific Gene Expression Cassette, Bacteria Containing the Same, and Use." The said prior application is incorporated into this application in its entirety by reference.

[0002] The present invention relates to a hypoxia-specific gene expression cassette, bacteria containing the same, and its use, and belongs to the fields of genetic engineering and biotherapy. [Background technology]

[0003] Solid tumors, such as sarcomas, melanomas, breast cancer, lung cancer, colon cancer, and prostate cancer, account for approximately 90% of all malignant tumors.

[0004] The tumor microenvironment of solid tumors shares common characteristics such as an abnormal tumor vascular system, excessive connective tissue, immunosuppression, an acidic environment, and hypoxic regions. Furthermore, the abnormal microenvironment of solid tumors acts as a natural barrier that makes it difficult for conventional therapeutic agents to penetrate, and chemotherapy drugs and antibody-based drugs have difficulty diffusing within the solid tumor microenvironment. In addition, due to a deficiency of oxygen free radicals, tumors develop resistance to chemotherapy and radiotherapy.

[0005] Due to the hypoxic environment within solid tumors, facultative and obligate anaerobic bacteria can invade the tumor and inhibit its growth, making them highly promising as therapeutic agents or vectors.

[0006] As early as 1868, German physician W. Busch first reported that in some tumor patients, bacterial infection (Streptococcus pneumoniae) suppressed tumor growth and, in some cases, completely eliminated the tumor. Thirty years later, American physician Coley and German physician Fehleisen each reported that bacterial infection could suppress tumors. These early studies were controversial because the results were difficult to reproduce and controlling bacterial toxicity was challenging. However, subsequent rigorous animal experiments demonstrated that bacterial infection reliably reduced tumor size and activated the host's immune system during treatment. In 1975, Carswell first reported that endotoxins (lipopolysaccharides) derived from Gram-negative bacteria could stimulate the immune system, promoting the release of tumor necrosis factor (TNF-α) and causing tumor cell death. Several bacterial vaccines have also been shown to treat tumors by stimulating the immune system. Among these, BCG vaccine (Bacillus Calmette-Guérin vaccine) was the first biological agent used in the clinical treatment of tumors. BCG is a suspension of attenuated live bacteria obtained by serially inoculating highly pathogenic Mycobacterium bovine tuberculosis for 230 generations, and is used for the prevention of tuberculosis. Numerous studies and clinical practice have confirmed that BCG is one of the most effective treatments for bladder cancer.

[0007] In recent years, with the rapid advancements in molecular biology and genetic engineering technologies, research has revealed that certain facultative or obligate anaerobic bacteria can target solid tumors, colonize and proliferate, and induce tumor regression. Examples include the obligate anaerobic bacterium Clostridium and the probiotic Bifidobacterium. Among these, the Gram-negative facultative anaerobic bacterium Salmonella enterica shows great potential for future applications.

[0008] Studies have shown that by weakening Salmonella typhi through various methods, its ability to colonize tumor tissue can be 1,000 to 10,000 times greater than that of normal tissue. Methods for weakening this strain include the following:

[0009] 1. aroA gene deficiency, bacterial species SL7207. The aroA mutant strain cannot synthesize para-aminobenzoic acid (PABA) and aromatic amino acids on its own, resulting in a lower PABA content in the in vivo environment and thus reduced toxicity.

[0010] 2. PurI and msbB gene deficiencies, bacterial strain VNP20009. Mutations in purI inhibit adenosine monophosphate synthesis, reducing toxicity in vivo. Mutations in msbB can inhibit the synthesis of lipid A in cell wall lipopolysaccharide, thereby avoiding bacterial sepsis caused by excessive TNF-α secretion. In preliminary animal studies, this strain was able to suppress tumor growth, and the safety of live bacteria administered intravenously was demonstrated in a Phase I clinical trial.

[0011] 3. Leucine and Arginine Deficiencies, Strains A1 and A1-R. Zhao et al. identified A1, which can inhibit tumor cell growth in both in vivo and in vitro experiments, through mutagenesis and screening using chemical reagents. To enhance its tumor-targeting ability, Zhao et al. re-isolated A1 from tumors and obtained the A1-R strain, which has a higher affinity for tumor tissue. Furthermore, Salmonella not only possesses the ability to inhibit tumor growth itself, but can also readily carry various DNA vectors. For example, E. coli cytosine deaminase (CD), mitomycin C, and colicin E3 were introduced into VNP20009, and therapeutic trials were conducted in various tumor models.

[0012] While the above nutritional requirements method can significantly reduce the distribution of bacteria in normal organs such as the liver and spleen, it is difficult to observe a significant decrease in the distribution of bacteria in normal organs over time.

[0013] Salmonella YB1 is a tumor-targeting strain developed by combining chromosome homologous recombination technology with the concepts of synthetic biology, based on previous research. It thrives in hypoxic areas of tumors and has the ability to accumulate within them.

[0014] The specific method involves controlling the expression of the lethal gene asd by combining the anaerobic promoter pepT and the reverse oxygen promoter sodA, thereby enabling tumor survival and growth under hypoxic conditions and reducing accumulation in normal organs. This improves safety compared to conventional oncolytic bacteria.

[0015] YB1 is a first-generation attenuated Salmonella strain based on an oxygen-regulating promoter designed through analysis of intratumor oxygen. Therefore, it is mechanistically significantly superior to typical trophication-dependent strains. However, due to the bacterium's susceptibility to mutation, it is necessary to monitor bacterial mutations under various stress conditions. In 2011, Dr. Yu Bin pointed out that YB1 evolves very readily in the absence of diaminopimelic acid (DAP), and as a result, escapes oxygen regulation. Studies have shown that the mutation frequency of YB1 in relation to oxygen can be up to 10 per bacterium. -5 ~10 -6 It can be seen that... On the other hand, according to research, the mutation frequency of Escherichia coli, which, like Salmonella, belongs to the Enterobacteriaceae genus, is 10 per generation. -9 ~10 -10 In other studies, the natural mutation rate of Salmonella is 10 per year. -8In contrast, the division rate of Salmonella has been shown to be 20-30 minutes per generation. The YB1 mutation frequency calculated from this is 10,000 times higher than the natural mutation frequency of Salmonella. Therefore, a significant increase in the YB1 mutation frequency is abnormal. This abnormally high frequency of mutations may reduce the benefits of hypoxia control, affect its safety, and potentially introduce new risks to tumor treatment. The underlying cause may be due to the irrational regulation of genetic elements such as the pepT promoter, asd gene, and sodA promoter. Therefore, the discovery of new regulatory mechanisms is essential.

[0016] A 2012 study on YB1 showed a decreasing trend in the distribution of YB1 in normal organs of mice, but it was difficult to completely eliminate it in the short term. Even 11 days after injection, the amount was still 10 per gram of tissue. 3 ~10 4 It was present. It took up to 26 days for it to be completely eliminated from most of the mouse's normal organs, and small amounts remained in the liver. This may be related to a rapid evolutionary rate caused by a design flaw in YB1 itself.

[0017] As described above, there is an urgent need in this field for a new generation of viable bacterial strains that can efficiently target tumors, rapidly remove them in normal organs, and effectively suppress tumor growth. [Overview of the project] [Problems that the invention aims to solve]

[0018] In this invention, unless otherwise specified, the scientific and technical terms used herein have meanings generally understood by those skilled in the art. Furthermore, the related terms and experimental procedures used in this invention in protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology are all terms and general steps widely used in their respective fields.

[0019] This invention provides a hypoxia-specific gene expression cassette to address the above-mentioned defects in the prior art. a) A promoter containing an FNR binding site, which is a forward hypoxic promoter whose expression can be induced under hypoxic conditions. b) Essential genes for survival, and c) A promoter comprising FNR and ArcA binding sites, comprising a reverse hyperoxygen promoter capable of functioning under conditions of normal organ oxygen content, The aforementioned survival-essential gene is the gene that codes for alanine racemase.

[0020] Another aspect of the present invention provides a hypoxia-specific gene expression cassette. a) A promoter containing an FNR binding site, which is a forward hypoxic promoter whose expression can be induced under hypoxic conditions. b) Essential genes for survival, and c) A promoter comprising two ArcA binding sites, comprising a reverse hyperoxygen promoter capable of functioning under conditions of normal organ oxygen content, The aforementioned survival-essential gene is the gene that codes for alanine racemase.

[0021] In this invention, "polypeptide," "peptide," and "protein" are interchangeable 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 corresponding natural amino acids, as well as polymers of natural amino acids. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include, but are not limited to, modified forms such as glycosylation, lipid binding, sulfated carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0022] In the present invention, "polynucleotide" refers to a polymer in which multiple nucleotides are linked by phosphate diester bonds, and the nucleotides include ribonucleotides and deoxyribonucleotides. The polynucleotide sequence of the present invention can be codon-optimized for various host cells (e.g., E. coli), thereby improving polypeptide expression. Methods for codon optimization are known in the art.

[0023] "Sequence identity" between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for evaluating the level of sequence identity between polypeptide sequences or polynucleotide sequences are known in the art. Sequence identity can be evaluated using various known sequence analysis software. For example, sequence identity can be evaluated using the online alignment tool EMBL-EBI (https: / / www.ebi.ac.uk / Tools / psa / ). Sequence identity between two sequences can be evaluated using the Needleman-Wunsch algorithm with default parameters.

[0024] For bacteria to survive only under low oxygen concentration conditions, the following three conditions must be met:

[0025] 1) Control the intensity of the upstream promoter and the occurrence of background leaks. If the oxygen sensor is partially leaked and expressed without binding to the upstream promoter, oxygen control becomes impossible, and as a result, bacteria can survive at any oxygen concentration. Conversely, if the upstream promoter is too weak, downstream gene expression cannot be initiated, and bacteria cannot survive at any oxygen concentration.

[0026] 2) Select the appropriate essential genes for survival. By selecting essential survival genes, it becomes possible to ensure bacterial death if these genes are not expressed, and to rapidly ensure bacterial survival if their expression is induced under hypoxic conditions.

[0027] 3) When oxygen levels switch between low and high oxygen, the upstream promoter rapidly initiates transcription, ensuring the survival of the modified bacteria by quickly synthesizing and expressing essential survival genes. However, under high oxygen conditions, the expression of essential survival genes does not begin, resulting in the death of the bacteria.

[0028] Accordingly, in the present invention, "hypoxia-specific gene expression cassette" refers to a DNA fragment capable of initiating the expression of an essential gene under hypoxic conditions, and includes an essential gene controlled by a hypoxia-inducible promoter, and may further include other regulatory elements necessary for the expression of said essential gene, if necessary.

[0029] In this invention, "essential gene" refers to a gene that plays a crucial role in bacterial growth and / or survival. If a bacterium lacks this gene or its functional expression, it cannot survive, divide, and / or grow normally. A typical example of a deficiency in an essential gene or its functional expression is a trophozoite strain. A trophozoite strain cannot survive, divide, and / or grow normally under in vitro or in vivo conditions without a specific exogenous supplement. Essential genes typically exist as a single copy on the bacterial chromosome.

[0030] Therefore, the requirements for essential genes for survival are as follows:

[0031] (1) This gene is essential for bacterial reproduction, and its deficiency leads to the rapid death of bacteria.

[0032] (2) Since this gene product does not exist in normal environments or the human body, it cannot escape control in the human environment. Furthermore, by adding the corresponding expression product of this gene to a normal culture environment, bacterial culture and preparation can be facilitated.

[0033] (3) This gene needs to be rapidly initiated under the control of a hypoxia promoter, and its product must be rapidly synthesized to perform a regulatory function in the host bacterium.

[0034] In the present invention, the survival-essential gene is a gene that encodes alanine racemase.

[0035] For Gram-negative bacteria such as E. coli and Salmonella, the cell wall is an essential component. The core component of the cell wall is peptidoglycan. Bacteria require D-alanine as an essential component for peptidoglycan synthesis. Without D-alanine, bacteria cannot synthesize the cell wall, leading to cell lysis.

[0036] In nature, all amino acids are in the L form. Therefore, Gram-negative bacteria have two genes, the alur gene and the dadX gene, which are responsible for the biosynthesis of alanine racemase. These genes play a role in converting L-alanine to D-alanine to meet the needs of cell wall synthesis.

[0037] The study revealed that simultaneous mutations in the arl gene and dadX gene can lead to lethal mutations in Salmonella bacteria, and that such mutations can be corrected by adding D-alanine to the culture medium.

[0038] Previous studies have shown that Salmonella YB1 is regulated by the asd gene as an essential gene. For Salmonella YB1, the present invention selects the arl gene and dadX gene as essential survival genes. The arl gene and dadX gene belong to the category of functionally homologous genes.Therefore, in the present invention, the alanine racemase gene is the alr gene of Salmonella (SEQ ID No. 17) and the protein sequence of Salmonella arl (SEQ ID No. 18), or the dadX gene of Salmonella (SEQ ID No. 19) and the protein sequence of Salmonella dadX (SEQ ID No. 20); or the alr gene of Escherichia coli (SEQ ID No. 21) and the protein sequence of Escherichia coli arl (SEQ ID No. 22), or the dadX gene of Escherichia coli (SEQ ID No. 23) and the protein sequence of Escherichia coli dadX (SEQ ID No. 24); or the alr gene of Shigella (SEQ ID No. 25) and the protein sequence of Shigella arl (SEQ ID No. 26), or the dadX gene of Shigella (SEQ ID No. 27) and the protein sequence of Shigella dadX (SEQ ID No. No. 28); or the alr gene of Klebsiella (SEQ ID No. 29) and the protein sequence of Klebsiella arr (SEQ ID No. 30), or the dadX gene of Klebsiella (SEQ ID No. 31) and the protein sequence of Klebsiella dadX (SEQ ID No. 32); or the alr gene of Yersinia (SEQ ID No. 33) and the protein sequence of Yersinia arr (SEQ ID No. 34), or the dadX gene of Yersinia (SEQ ID No. 35) and the protein sequence of Yersinia dadX (SEQ ID No. 36); or the alr gene of Haemophilus (SEQ ID No. 37) and the protein sequence of Haemophilus arr (SEQ ID No. 38); or the alr gene of Pseudomonas (SEQ ID No. 39) and the protein sequence of Pseudomonas arr (SEQ ID It may also be derived from No. 40), or from the dadX gene of Pseudomonas (SEQ ID No. 41) and the Pseudomonas dadX protein sequence (SEQ ID No. 42).

[0039] In the present invention, the alur gene and dadX gene of the alanine racemase contain nucleotide sequences having sequence identity of 81% or more, preferably 82% or more, more preferably 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or most preferably 99% or more, with respect to the specific sequences described above.

[0040] In this invention, bacteria with the alfr gene knocked out were first constructed, and then another gene, dadX, was modified to create bacteria in which both the alfr and dadX genes were deficient after editing. In addition, the YB1 forward hypoxia promoter pepT and reverse hyperxia promoter sodA were used to control an additional alfr gene (SWT006). According to the experimental results (SWT006 verification experiment), it was demonstrated that selecting the alfr and dadX genes as essential gene control means yielded higher controllability than the essential gene asd previously selected in YB1 Salmonella.

[0041] The forward hypoxia promoter described in this invention is a hypoxia condition promoter regulated by FNR, and the reverse hyperxia promoter described in this invention is an antisense promoter negatively regulated by FNR and / or ArcA. The fumarate and nitrate reduction gene fnr is a key gene that regulates aerobic and anaerobic growth in Salmonella, and this complex regulatory system has been widely studied in Escherichia coli and Salmonella. The DNA-binding protein FNR encoded by the fnr gene senses changes in oxygen, controls the expression of different genes, and enables switching at the overall metabolic level. Therefore, DNA-binding sequences such as FNR and ArcA are important ways of controlling the expression of downstream genes.

[0042] FNR is oxygen-sensitive [4Fe-4S] 2+It possesses a domain and can directly sense oxygen, regulating site-specific DNA binding. On the other hand, ArcA senses signals from the aerobic respiratory chain. Therefore, Gram-negative facultative anaerobic bacteria such as Salmonella and E. coli have two different mechanisms for sensing changes in oxygen concentration. When FNR controls gene expression under hypoxic conditions, there are two types: activation and repression.

[0043] Therefore, the present invention screened for forward hypoxic promoters, yhbU, ynfK, yecH, cydA, focA, and tdcA, in which FNR activates downstream gene expression under hypoxic conditions, and reverse hyperxic promoters, ydcI, cyoA, phoH, argT, mqo, and lldP, in which FNR and / or ArcA suppress downstream gene expression under hypoxic conditions. Furthermore, promoters from different origins were screened to be used as forward hypoxic and reverse hyperxic promoters in the present invention.

[0044] The forward hypoxic promoter used in this invention is as follows: (1) Salmonella yhbU promoter (yhbU-S) (SEQ ID No. 1): The FNR binding site contained in it is "CTGCCTTAAATCAA". (2) Escherichia coli ynfK promoter (ynfK-E) (SEQ ID No. 2): The FNR binding site contained in it is "TTGCGCTATCTCAA". (3) Salmonella tdcA promoter (tdcA-S) (SEQ ID No. 3): The FNR binding site contained in it is "TTGATTGAAATCAG". (4) Escherichia coli tdcA promoter (tdcA-E) (SEQ ID No. 4): The FNR binding site contained in it is "TTGACAAAAATCAG". (5) E. coli yecH promoter (yecH-E) (SEQ ID No. 5): The FNR binding site contained in it is "TTCATAAGCGGCAA". (6) Salmonella ynfK promoter (ynfK-S) (SEQ ID No. 6): The FNR binding site contained in it is "TTGCGCTAACTCAA". (7) Escherichia coli cydA promoter (cydA-E) (SEQ ID No. 7): The FNR binding site contained in it is "TTGATATTTATCAA". (8) Escherichia coli focA promoter (focA-E) (SEQ ID No. 8): The FNR binding site contained in it is "ATGATCTATATCAA". The reverse-direction high-oxygen promoter used in this invention is as follows. (1) Salmonella cyoA promoter (cyoA-S) (SEQ ID No. 9): The FNR binding site contained in it is "TTTATTGATAATAA", and the ArcA binding site is "GTTAAGTA". (2) Salmonella phoH promoter (phoH-S) (SEQ ID No. 10): The FNR binding site contained in it is "TTGAGCGCCGTTAA", and the ArcA binding site is "GTTAATTA". (3) Salmonella lldP ​​promoter (lldP-S) (SEQ ID No. 11): The binding site for FNR contained in it is "TCCATACACAACAA", and the binding site for ArcA is "GTTAACTA". (4) Salmonella argT promoter (argT-S) (SEQ ID No. 12): It contains two ArcA binding sites, the first ArcA binding site being "TTTAATTC" and the second ArcA binding site being "TTTTATTA". (5) Salmonella ydcI promoter (ydcI-S) (SEQ ID No. 13): The binding site for FNR contained in it is "GTTATCAAAAACAA", and the binding site for ArcA is "GTTAATAA". (6) Escherichia coli phoH promoter (phoH-E) (SEQ ID No. 14): The FNR binding site contained in it is "TTCATCACTGTCAT", and the ArcA binding site is "GTTAAATA". (7) Escherichia coli lldP ​​promoter (lldP-E) (SEQ ID No. 15): The FNR binding site contained in it is "TTCATTGTCATTAT", and the ArcA binding site is "TTTAGTTA". (8) Escherichia coli ydcI promoter (ydcI-E) (SEQ ID No. 16): The FNR binding site contained in it is "GTTATCATAATCAA", and the ArcA binding site is "GTTAATAA". (9) Escherichia coli MQO promoter (MQO) (SEQ ID No. 62): It contains two ArcA binding sites, the first ArcA binding site being "ATTATTTA" and the second ArcA binding site being "GTTACTTA".

[0045] Analysis of the reverse hypoxia promoter and forward hyperxia promoter described above revealed that all FNR binding sites conform to the pattern "TTGATNNNNATCAA". Furthermore, any base in the TTGAT and ATCAA sequences of the conserved binding site is substituted, but the total number of substitutions is 3 or less, and it is not possible to substitute three consecutive bases. ArcA conforms to the pattern "GTTAATTA" in its core region, and any base in the GTTAATTA sequence of the conserved binding site is substituted, but the total number of substitutions is 2 or less.

[0046] Therefore, in a preferred embodiment of the present invention, the FNR binding site of the forward hypoxia promoter or the reverse hyperxia promoter conforms to the pattern TTGATNNNNATCAA, where N is one of the bases A, T, C, or G. Any base in the TTGAT or ATCAA sequence of the conservative binding site is substitutable, but the total number of substitutions is 3 or less, and three consecutive bases cannot be substituted. The ArcA binding site of the reverse hyperxia promoter conforms to the pattern GTTAATTA, where any base is substitutable, but the total number of substitutions is 2 or less.

[0047] In a more preferred embodiment of the present invention, the forward hypoxia promoter is selected from yhbU, ynfK, tdcA, yecH, or focA, the survival essential gene is selected from alr or dadX, and the reverse hyperxia promoter is selected from cyoA, ydcI, phoH, argT, mqo, or lldP.

[0048] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene arl, and a reverse hyperxia promoter cyoA.

[0049] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene alur, and a reverse hyperxia promoter ydcI.

[0050] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene arl, and a reverse hyperxia promoter phoH.

[0051] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene arl, and a reverse hyperxia promoter argT.

[0052] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene alur, and a reverse hyperxia promoter mqo.

[0053] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter ynfK, the survival essential gene arl, and the reverse hyperxia promoter cyoA.

[0054] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene arlr, and a reverse hyperxia promoter phoH.

[0055] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter ynfK, the survival essential gene arl, and the reverse hyperxia promoter argT.

[0056] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter ynfK, the survival essential gene alur, and the reverse hyperxia promoter ydcI.

[0057] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene alur, and a reverse hyperxia promoter mqo.

[0058] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter tdcA, a survival essential gene alur, and a reverse hyperxia promoter ydcI.

[0059] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter tdcA, a survival essential gene arlr, and a reverse hyperxia promoter cyoA.

[0060] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter yecH, the survival essential gene arlr, and the reverse hyperxia promoter cyoA.

[0061] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter yecH, the survival essential gene arlr, and the reverse hyperxia promoter lldP.

[0062] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene alfr, and a reverse hyperxia promoter argT.

[0063] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene arlr, and a reverse hyperxia promoter phoH.

[0064] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene alur, and a reverse hyperxia promoter mqo.

[0065] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter focA, a survival essential gene arlr, and a reverse hyperxia promoter lldP.

[0066] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter cyoA.

[0067] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter ydcI.

[0068] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter phoH.

[0069] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter argT.

[0070] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter mqo.

[0071] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter ynfK, the survival essential gene dadX, and the reverse hyperxia promoter cyoA.

[0072] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperxia promoter phoH.

[0073] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter ynfK, the survival essential gene dadX, and the reverse hyperxia promoter argT.

[0074] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter ynfK, the survival essential gene dadX, and the reverse hyperxia promoter ydcI.

[0075] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperxia promoter mqo.

[0076] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter tdcA, a survival essential gene dadX, and a reverse hyperxia promoter ydcI.

[0077] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter tdcA, a survival essential gene dadX, and a reverse hyperxia promoter cyoA.

[0078] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter yecH, the survival essential gene dadX, and the reverse hyperxia promoter cyoA.

[0079] In a more preferred embodiment of the present invention, the expression cassette comprises the forward hypoxia promoter yecH, the survival essential gene dadX, and the reverse hyperxia promoter lldP.

[0080] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene dadX, and a reverse hyperxia promoter argT.

[0081] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene dadX, and a reverse hyperxia promoter phoH.

[0082] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene dadX, and a reverse hyperxia promoter mqo.

[0083] In a more preferred embodiment of the present invention, the expression cassette comprises a forward hypoxia promoter focA, a survival essential gene dadX, and a reverse hyperxia promoter lldP.

[0084] The forward hypoxia promoter disclosed in patent CN104471057B is pepT. The pepT promoter is not a promoter fully regulated by FNR, but a dual-regulated promoter of CRP-cAMP and FNR. Thus, half of the FNR binding site of the pepT promoter is a CRP binding site, and the other half is an FNR binding site. The CRP-FNR binding region sequence of the pepT promoter is GTGACCTGACGCAA, where the first half GTGA corresponds to the first half of the CRP-conservative binding site GTGANNNNNNTCAC, while the second half CGCAA corresponds to part of the FNR-conservative region ATCAA. The 10th A is substituted with C, and the 11th T is substituted with G, which does not conform to the rules for forward hypoxia promoters in the present invention.

[0085] The FNR binding region TTGATAATCATTTT of the reverse hyperoxygen promoter sodA disclosed in patent CN104471057B includes only the first half of the FNR, TTGAT, and the latter half does not include the complete FNR binding site because three consecutive ATCAA are replaced with ATTTT. The ArcA binding site TTTAATTA is included, but the first G is replaced with T compared to the core-conservative binding site 5'-GTTAATTA-3' of ArcA. Therefore, the sodA promoter contains only one ArcA binding site and the FNR binding site is incomplete, which does not conform to the rules for reverse hyperoxygen promoters of the present invention.

[0086] As described above, the present invention provides a more efficient and safer hypoxia-specific gene expression cassette by screening for rational forward hypoxia promoters and reverse hyperxia promoters and combining them with appropriate survival essential genes.

[0087] In a preferred embodiment of the present invention, the forward hypoxic promoter and / or reverse hyperxic promoter are promoters derived from Gram-negative bacteria.

[0088] In this invention, the term "Gram-negative bacteria" refers to bacteria that, after performing a portion of a known Gram staining method, do not retain the initial basic dye (e.g., crystal violet) as a staining agent. In an exemplary Gram staining, cells are first fixed onto a glass slide by heating and stained with a basic dye (e.g., crystal violet) absorbed by both Gram-negative and Gram-positive bacteria. Next, the glass slide is treated with a mordant (e.g., Gram iodine solution). The mordant binds to the basic dye (e.g., crystal violet) and is captured within the cells. Next, the cells are washed with acetone or ethanol and counterstained with a second dye of a different color (e.g., safranin). Gram-positive organisms retain the initial purple stain, while Gram-negative organisms are decolorized by the organic washing solvent, thus exhibiting counterstaining. Examples of Gram-negative bacteria include, but are not limited to, species of Escherichia coli, Shigella, Salmonella, Campylobacter, Neisseria, Haemophilus, Aeromonas, Francisella, certain species of Yersinia, certain species of Klebsiella, certain species of Bordetella, Legionella, Corynebacterium, Citrobacter, Chlamydia, Brucella, Pseudomonas, Helicobacter, and Vibrio.

[0089] Therefore, in a more preferred embodiment of the present invention, Gram-negative bacteria include Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Chronobacter, Klebsiella, Pantoea, Serratia, Simwellia, Enterobacter ludwigia, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Bordetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, and Intestinal bacteria. The selection includes Vibrio parahaemolyticus, Vibrio cholerae, Plague bacillus, Neisseria catarrhagicum, Moraxella catarrhalis, Campylobacter jejuni, Shigella, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Plague bacillus, Bacillus sonne, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori.

[0090] In this invention, the term "live bacteria" refers to a bacterial strain that possesses viability, active nutrient metabolic characteristics, and is capable of performing its own biological functions. Live bacteria also include the bacterial biomass produced during the metabolic processes of the bacterial strain.

[0091] In a more preferred embodiment of the present invention, the forward hypoxia promoter is yhbU selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Chronobacter, Klebsiella, Pantoea, Serratia, and Simwellia.

[0092] In a more preferred embodiment of the present invention, the forward hypoxia promoter is ynfK selected from Salmonella, Escherichia coli, Serratia, Shigella, and Enterobacter dulcis.

[0093] In preferred embodiments of the present invention, the alanine racemase gene alur and / or dadX is selected from Salmonella, Escherichia coli, Shigella, Klebsiella, Yersinia, Haemophilus, and Pseudomonas.

[0094] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is controlled by the oxygen concentration.

[0095] For Gram-negative bacteria such as Salmonella to survive in hypoxic regions, an oxygen concentration of 1% is a crucial threshold indicating pathological hypoxia. This is because in tumors, a hypoxic region is clearly defined when the oxygen concentration falls below 1%. Normal organs and tissues do not have regions with oxygen concentrations below 1%. For example, in various tumors such as pancreatic cancer, cervical cancer, and prostate cancer, the hypoxic region is characterized by an oxygen concentration of less than 0.7%.

[0096] The hypoxia-specific gene expression cassette designed in this invention allows Gram-negative bacteria such as Salmonella to recognize hypoxic regions in tumors and proliferate in those regions by precisely controlling oxygen concentration. The objective of this invention is to enable modified Gram-negative bacteria such as Salmonella to survive and proliferate when the oxygen concentration is less than 0.8%, and to undergo cytotoxic lysis in environments with normal oxygen concentrations.

[0097] Therefore, in a more preferred embodiment of the present invention, the forward hypoxia promoter functions when the oxygen content is less than 1%, but does not function when the oxygen content is greater than 1% and / or

[0098] The reverse high-oxygen promoter works when the oxygen content is above 1%, but it cannot work when the oxygen content is below 1%.

[0099] In a more preferred embodiment of the present invention, the forward hypoxia promoter functions when the oxygen content is less than 0.8%, but does not function when the oxygen content is greater than 0.8% and / or The reverse high-oxygen promoter works when the oxygen content is above 0.8%, but does not work when the oxygen content is below 0.8%.

[0100] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is incorporated into the chromosome of the host bacterium.

[0101] In another aspect of the present invention, modified Gram-negative bacteria are provided, modified with the hypoxia-specific gene expression cassette described in the present invention.

[0102] In preferred embodiments of the present invention, the Gram-negative bacteria include Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Chronobacter, Klebsiella, Pantoea, Serratia, Simwellia, Enterobacter ludwigia, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Bordetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, and enteritis. These include Vibrio, Vibrio cholerae, Plague bacillus, Neisseria catarrhagicum, Moraxella catarrhalis, Campylobacter jejuni, Shigella, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Plague bacillus, Bacillus sonne, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori.

[0103] In a more preferred embodiment of the present invention, the Gram-negative bacterium is Salmonella typhi.

[0104] Another aspect of the present invention provides the use of a hypoxia-specific gene expression cassette or Gram-negative bacteria as described in the present invention in the preparation of an antitumor drug.

[0105] In a preferred embodiment of the present invention, the tumor is a solid tumor.

[0106] As used in this invention, the term "solid tumor" refers to an abnormal mass of tissue, which typically does not include cysts or fluid areas. Solid tumors can be either benign (non-cancerous) or malignant (cancerous). Types of malignant solid tumors are named after the types of cells that form them. Examples of malignant solid tumors include sarcomas, cancers, and lymphomas. Leukemia (blood cancer) does not typically form malignant solid tumors. Malignant solid tumors include, but are not limited to, abnormal cell masses that may originate from a variety of tissue types, such as the liver, colon, colorectal rectum, skin, mammary glands, pancreas, cervix, uterine body, bladder, gallbladder, kidneys, larynx, lips, oral cavity, esophagus, ovaries, prostate, stomach, testes, thyroid gland, and lungs. Therefore, malignant solid tumors include malignant solid liver tumors, colon tumors, colorectal tumors, skin tumors, breast cancer, pancreatic tumors, cervical tumors, uterine body tumors, bladder tumors, gallbladder tumors, kidney tumors, laryngeal tumors, lip tumors, oral tumors, esophageal tumors, ovarian tumors, prostate tumors, gastric tumors, testicular tumors, thyroid tumors, lung tumors, and others.

[0107] Therefore, in a more preferred embodiment of the present invention, the solid tumor is selected from tumors / cancers such as breast cancer, bone cancer, liver cancer, lung cancer, skin cancer, kidney cancer, stomach cancer, pancreatic cancer, prostate cancer, lymph node cancer (non-Hodgkin lymphoma, Hodgkin lymphoma), intestinal cancer (colon cancer, rectal cancer), pelvic cavity cancer (cervical cancer, malignant ovarian tumor, endometrial cancer, ovarian cancer), nervous system cancer, head and neck cancer, and bladder cancer.

[0108] In a more preferred embodiment of the present invention, 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.

[0109] In the present invention, the term "subject" preferably refers to a subject requiring treatment for a target condition such as a tumor, when used for therapeutic purposes. When used for preventive purposes, the subject is preferably a subject at risk of developing a target condition or prone to developing a target condition. The term "subject" includes organisms such as prokaryotes and eukaryotes. Examples of subjects include mammals such as humans, dogs, cattle, horses, pigs, sheep, goats, cats, mice, rabbits, hedgehogs, and rats, and transgenic non-human animals. In certain embodiments of the present invention, the subject is a human.

[0110] As used in the present invention, “treatment” means a process for obtaining a beneficial or desirable clinical outcome. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, one or more of the following: reduction of the proliferation of neoplastic or cancerous cells (or destruction of neoplastic or cancerous cells), inhibition of metastasis of neoplastic cells, reduction or decrease in tumor size, relief of malignancy, reduction of symptoms caused by malignancy, improvement of the quality of life of patients with malignancy, reduction of the dosage of other drugs required to treat malignancy, delay of malignancy progression, cure of malignancy, and / or extension of survival of patients with malignancy.

[0111] As used in the present invention, an "effective amount" or "effective dose" of a bacterium, drug, or pharmaceutical composition is an amount sufficient to achieve one or more beneficial or desired results. For prophylactic use, beneficial or desired results include elimination or reduction of the risk of a disease, reduction of the severity of a disease, or delay of the onset of a disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the progression of the disease. For therapeutic use, beneficial or desired results include, for example, alleviation of one or more symptoms of a disease (e.g., a tumor), reduction of the dosage of other drugs required for the treatment of the disease, enhancement of the effect of other drugs, prolongation of the survival period of the subject to be treated, and / or delay of 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%, compared to a subject not receiving treatment. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts the effectiveness of a treatment on human tumors. Alternatively, the effectiveness can be evaluated by examining the ability to inhibit cell growth. Such inhibition can be measured in vitro using assays known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate the symptoms of a subject. Those skilled in the art can determine this amount based on factors such as the age of the subject, the severity of the subject's symptoms, the specific composition selected or the route of administration.

[0112] In some embodiments of the present invention, the effective amount of bacteria in said bacterium, drug or pharmaceutical composition is at least about 10 4 colony forming units (cfu), for example, at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 1011 , 10 12 or 10 13 It includes cfu, which is a range between any enumerated values, for example, 10 4 ~10 8 cfu, 10 4 ~10 9 cfu, 10 4 ~10 10 cfu, 10 4 ~10 11 cfu, 10 4 ~10 12 cfu, 10 4 ~10 13 cfu, 10 5 ~10 8 cfu, 10 5 ~10 9 cfu, 10 5 ~10 10 cfu, 10 5 ~10 11 cfu, 10 5 ~10 12 cfu, 10 5 ~10 13 cfu, 10 6 ~10 8 cfu, 10 6 ~10 9 cfu, 10 6 ~10 10 cfu, 10 6 ~10 11 cfu, 10 6 ~10 12 cfu, 10 6 ~10 13 cfu, 10 7 ~10 8 cfu, 10 7 ~10 9 cfu, 10 7 ~10 10 cfu, 10 7 ~10 11 cfu, 10 7 ~10 12 cfu, 10 7 ~10 13 cfu, 10 8 ~10 9 cfu, 10 8 ~10 10 cfu, 10 8~10 11 cfu, 10 8 ~10 12 cfu, or 10 8 ~10 13 Includes CFU.

[0113] In some embodiments of the present invention, the volume of an effective dose of the bacteria, drug, or pharmaceutical composition of the present invention is about 3 ml or less, about 2.5 ml, about 2 ml, about 1.5 ml, about 1 ml, about 0.75 ml, about 0.5 ml, about 0.25 ml, or about 0.1 ml. In some embodiments, the volume of an effective dose of the bacteria, drug, or pharmaceutical composition is about 20 ml, about 19 ml, about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml, about 13 ml, about 12 ml, about 11 ml, about 10 ml, about 9 ml, about 8 ml, about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml, or about 1 ml. Optionally, the volume of the effective dose of the bacteria, drug, or pharmaceutical composition is approximately 20.5 ml, approximately 19.5 ml, approximately 18.5 ml, approximately 17.5 ml, approximately 16.5 ml, approximately 15.5 ml, approximately 14.5 ml, approximately 13.5 ml, approximately 12.5 ml, approximately 11.5 ml, approximately 10.5 ml, approximately 9.5 ml, approximately 8.5 ml, approximately 7.5 ml, approximately 6.5 ml, approximately 5.5 ml, approximately 4.5 ml, approximately 3.5 ml, approximately 2.5 ml, approximately 1.5 ml, or approximately 0.5 ml. In some embodiments, the volume of the effective dose of the bacteria, drug, or pharmaceutical composition is approximately 200 ml, approximately 190 ml, approximately 180 ml, approximately 170 ml, approximately 160 ml, approximately 150 ml, approximately 140 ml, approximately 130 ml, approximately 120 ml, approximately 110 ml, approximately 100 ml, approximately 90 ml, approximately 80 ml, approximately 70 ml, approximately 60 ml, approximately 50 ml, approximately 40 ml, or approximately 30 ml, and a range between any of the listed values, for example, 100 ml to 110 ml, 100 ml to 120 ml, 90 ml to 120 ml, 90 ml to 130 ml, etc. Optionally, the volume of the effective dose of the bacteria, drug, or pharmaceutical composition is approximately 205 ml, approximately 195 ml, approximately 185 ml, approximately 175 ml, approximately 165 ml, approximately 155 ml, approximately 145 ml, approximately 135 ml, approximately 125 ml, approximately 115 ml, approximately 105 ml, approximately 95 ml, approximately 85 ml, approximately 75 ml, approximately 65 ml, approximately 55 ml, approximately 45 ml, approximately 35 ml, or approximately 25 ml, and a range between any of the listed values, for example, 105 ml to 115 ml, 105 ml to 125 ml, 95 ml to 125 ml, 95 ml to 135 ml, etc.Optionally, the volume of the effective dose of the bacteria, drug, or pharmaceutical composition is approximately 900 μL, approximately 800 μL, approximately 700 μL, approximately 600 μL, approximately 500 μL, approximately 400 μL, approximately 300 μL, approximately 200 μL, or approximately 100 μL, or optionally approximately 950 μL, approximately 850 μL, approximately 750 μL, approximately 650 μL, approximately 550 μL, approximately 450 μL, approximately 350 μL, approximately 250 μL, approximately 150 μL, or approximately 50 μL.

[0114] In some embodiments, the volume of the effective dose of the bacteria, drug, or pharmaceutical composition is about 2.0 ml or less.

[0115] The dosage ranges discussed herein are illustrative and do not limit the range of dosages that a physician may choose. The amount of the active ingredient (e.g., the bacteria of the present invention) in the pharmaceutical composition of the present invention may vary depending on factors including the individual's medical condition, age, sex, weight, presence or absence of tumors, type of tumor being treated, severity of tumor, activity or survival rate of the bacteria, drug or pharmaceutical composition, route of administration, duration of treatment, any drugs used in combination with the bacteria, drug or pharmaceutical composition, diet and overall health status, and similar factors well known in the art. The administration regimen can be adjusted to obtain the optimal therapeutic response. For example, it may be administered as a single dose, in multiple doses over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation.

[0116] In the present invention, “pharmaceutically acceptable carrier” means any material, including but not limited to disintegrants, binders, fillers, buffers, tensioners, stabilizers, antioxidants, surfactants, or lubricants, that, when combined with the active ingredient, maintain the biological activity of the ingredient and do not react with the target immune system. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., injection or infusion). For example, depending on the route of administration, the bacteria of the present invention may be encapsulated with a material to protect the bacteria from acids or other natural conditions that may inactivate them. pharmaceutically acceptable carriers include physiological saline, PBS buffer, sterile aqueous solutions or dispersions, and powders for preparing injectable solutions or dispersions at time of use. The use of media and reagents for these pharmaceutically active substances is well known in the art. Conventional media or reagents may be used in the pharmaceutical compositions of the present invention, except to the extent that they are incompatible with the active compound.

[0117] Accordingly, the present invention provides a pharmaceutical composition comprising an effective amount of the hypoxia-specific gene expression cassette of the present invention or the modified bacteria of the present invention. In one embodiment, the modified bacteria are live bacteria.

[0118] In a preferred embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0119] In a more preferred embodiment of the present invention, the pharmaceutically acceptable carrier is selected from disintegrants, binders, fillers, buffers, tensioners, stabilizers, antioxidants, surfactants, and lubricants.

[0120] In a preferred embodiment of the present invention, the pharmaceutical composition is used for the treatment of solid tumors. The bacteria, drugs, or pharmaceutical compositions of the present invention may be administered by routes such as intravenous injection, intratumor injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0121] In preferred embodiments of the present invention, the bacteria, drugs, or pharmaceutical compositions of the present invention can be formulated into forms of administration via routes such as intravenous injection, intratumor injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0122] The dosage form of the drug or pharmaceutical composition of the present invention may be in the form of a solution, emulsion, lyophilized preparation, or suspension; for oral administration, the dosage form may be in the form of a tablet or capsule; for intranasal administration, the dosage form may be in the form of a powder, nasal spray, or aerosol; for topical administration, 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.

[0123] The bacteria, drugs, or pharmaceutical compositions of the present invention can be produced by methods well known in the art, such as microbial growth in a fermenter, followed by centrifugation and concentration, washing, filtration or dialysis, conventional granulation, mixing, dissolution, encapsulation, freeze-drying or emulsification processes, and other methods. The bacteria, drugs, or pharmaceutical compositions of the present invention can be produced in various forms, such as granules, precipitates or fine particles, powders including freeze-dried, tumble-dried or spray-dried powders, amorphous powders, injections, emulsions, elixirs, suspensions, or solutions. The formulations may optionally contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0124] 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 preferred embodiments of the present invention, the bacteria, drugs, or pharmaceutical compositions can be administered in combination with other malignant tumor treatments (including, but not limited to, radiotherapy, chemotherapy, and surgery). In such cases, the bacteria, drugs, or pharmaceutical compositions can be used as adjuvants.

[0125] In a further embodiment, the present invention provides a method for treating a tumor, comprising administering to a subject a hypoxia-specific gene expression cassette described in the present invention, a Gram-negative bacterium described in the present invention, or a pharmaceutical composition described in the present invention.

[0126] In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to treat a tumor. In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to induce an antitumor-specific immune response in a tumor-affected subject. In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to induce antitumor immunological memory in a tumor-affected subject. In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to prevent or treat tumor metastasis or recurrence. In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to treat tumors that have developed resistance to or have failed to treat previous antitumor therapies.

[0127] In one embodiment, the tumor is a tumor of the nervous system, respiratory system, digestive system, urinary system, reproductive system, hematopoietic system, lymphatic system, endocrine system, or skin or mucous membrane. In one embodiment, the tumor is a sarcoma or cancer. In one embodiment, the tumor is a solid tumor.

[0128] In a more preferred embodiment of the present invention, the solid tumor is selected from tumors / cancers such as breast cancer, bone cancer, liver cancer, lung cancer, skin cancer, kidney cancer, stomach cancer, pancreatic cancer, prostate cancer, lymph node cancer (non-Hodgkin lymphoma, Hodgkin lymphoma), intestinal cancer (colon cancer, rectal cancer), pelvic cavity cancer (cervical cancer, ovarian malignant tumor, endometrial cancer, ovarian cancer), nervous system cancer, head and neck cancer, and bladder cancer.

[0129] In a further preferred embodiment of the present invention, 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.

[0130] A method for treating tumors according to the present invention includes administering an effective amount of the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor.

[0131] In embodiments of the present invention, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention can be administered by routes such as 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.

[0132] In some embodiments of the present invention, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention may be administered at intervals of, for example, about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, and it may be administered at intervals of, for example, between any two of the listed values, e.g., 1 minute to 10 minutes. 1 minute to 30 minutes, 1 minute to 1 hour, 1 minute to 2 hours, 1 minute to 4 hours, 1 minute to 12 hours, 1 minute to 18 hours, 1 minute to 1 day, 10 minutes to 30 minutes, 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 4 hours, 10 minutes to 12 hours, 10 minutes to 18 hours, 10 minutes to 1 day, 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 4 hours, 30 minutes to 12 hours, 30 minutes to 18 hours, 30 minutes to 1 day , including 30 minutes to 2 days, 1 hour to 2 hours, 1 hour to 4 hours, 1 hour to 12 hours, 1 hour to 18 hours, 1 hour to 1 day, 4 hours to 12 hours, 4 hours to 18 hours, 4 hours to 1 day, 1 day to 2 days, 1 day to 3 days, 1 day to 4 days, 1 day to 5 days, 1 day to 7 days, 1 day to 10 days, 2 days to 3 days, 2 days to 4 days, 2 days to 5 days, 2 days to 7 days, 2 days to 10 days, or 5 days to 10 days. In some embodiments, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition may be administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks, or once every twenty-six weeks.In some embodiments, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is formulated to be administered at intervals of, for example, about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, and it is formulated to be administered at intervals of, for example, 1 minute to 10 minutes, 1 minute ~30 minutes, 1 minute to 1 hour, 1 minute to 2 hours, 1 minute to 4 hours, 1 minute to 12 hours, 1 minute to 18 hours, 1 minute to 1 day, 10 minutes to 30 minutes, 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 4 hours, 10 minutes to 12 hours, 10 minutes to 18 hours, 10 minutes to 1 day, 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 4 hours, 30 minutes to 12 hours, 30 minutes to 18 hours, 30 minutes to 1 day, The durations include 30 minutes to 2 days, 1 hour to 2 hours, 1 hour to 4 hours, 1 hour to 12 hours, 1 hour to 18 hours, 1 hour to 1 day, 4 hours to 12 hours, 4 hours to 18 hours, 4 hours to 1 day, 1 day to 2 days, 1 day to 3 days, 1 day to 4 days, 1 day to 5 days, 1 day to 7 days, 1 day to 10 days, 2 days to 3 days, 2 days to 4 days, 2 days to 5 days, 2 days to 7 days, 2 days to 10 days, or 5 days to 10 days. In some embodiments, the bacteria, drug, or pharmaceutical composition is formulated to be administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 15 weeks, once every 20 weeks, once every 25 weeks, or once every 26 weeks. The dosing regimen will vary depending on the pharmacokinetic decay pattern predicted by the physician. Treatment progress can be monitored using standard techniques and assays. The dosing regimen may change over time.

[0133] The present invention further provides a method for inducing an antitumor-specific immune response in a subject suffering from a tumor, which comprises administering an effective amount of the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention to the subject suffering from a tumor.

[0134] The present invention further provides a method for inducing antitumor immunological memory in a tumor-affected subject, comprising administering an effective amount of the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention to the tumor-affected subject.

[0135] The present invention further provides a method for preventing or treating tumor metastasis or recurrence, which comprises administering an effective amount of the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor.

[0136] The present invention further provides a method for preventing or treating tumor metastasis or recurrence, comprising administering an effective amount of the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject whose tumor has metastasized or recurred, or to a subject at high risk of tumor metastasis or recurrence.

[0137] The present invention further provides a method for treating tumors that have developed resistance to or have failed to treat prior antitumor therapies, comprising administering an effective amount of the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor that has developed resistance to or has failed to treat prior antitumor therapies. [Effects of the Invention]

[0138] By employing the above-described technical solution, the present invention achieves the following beneficial effects.

[0139] 1. In this invention, a more efficient and safer hypoxia-specific gene expression cassette is obtained by rationally designing a forward hypoxia promoter and a reverse hyperxia promoter and combining them with appropriate survival essential genes.

[0140] 2. In this invention, the alfr gene or the dadX gene is selected as the essential gene for survival. The regulatory capacity of the alfr gene and dadX gene, which are designated as essential genes for survival, is higher than that of the asd gene, an essential gene selected by Salmonella YB1.

[0141] 3. The hypoxia-specific gene expression cassette designed in this invention precisely controls oxygen, enabling modified bacteria to recognize hypoxic regions of tumors and proliferate in those regions. Even at oxygen concentrations below 0.8%, the modified bacteria modified in this invention can survive and proliferate, and are able to undergo cytotoxic lysis in environments with normal oxygen concentrations.

[0142] 4. Bacteria modified with the hypoxia-specific gene expression cassette designed in this invention exhibit a lower overall mutation rate. In a mouse in vivo tumor model, the modified bacteria are more widely distributed within the tumor and are more rapidly eliminated from normal organs. [Brief explanation of the drawing]

[0143] [Figure 1] This is a verification diagram of the identification of forward hypoxia promoter clone libraries by agarose gel electrophoresis. (A) Forward hypoxia promoters: yhbU-S, tdcA-S, ynfK-S, ynfk-E, tdcA-E, yecH-E, cydA-E, and focA-E (B) Forward hypoxia promoter yhbU-E [Figure 2] This is an agarose gel electrophoresis map for validation and amplification of essential gene libraries. [Figure 3] This is a verification diagram of the identification of reverse hyperoxygen promoter clone libraries by agarose gel electrophoresis. (A) Reverse hyperoxygen promoters cyoA-S, phoH-S, lldP-S, argT-S, ydcI-S, phoH-E, lldP-E, ydcI-E (B) Reverse hyperoxygen promoter mqo. [Figure 4] This is a schematic diagram of a hypoxia-specific gene expression cassette. [Figure 5] This is a schematic diagram of the lambda RED recombinase and CRE recombinase systems. [Figure 6] These are identification diagrams of the aroA gene in Salmonella obtained by knockout experiments. (A) This is a verification diagram of the identification of the aroA gene knockout fragment by agarose gel electrophoresis of the PCR amplification product. (B) This is a verification diagram of the identification of the target fragment of strain SWT003 by agarose gel electrophoresis of the PCR amplification product. [Figure 7] These are identification diagrams obtained from knockout experiments of Salmonella alr. (A) This is a verification diagram of identification by agarose gel electrophoresis of PCR amplification products of alr gene knockout fragments. (B) This is a verification diagram of identification by agarose gel electrophoresis of PCR amplification products of target fragments of strain SWT004. [Figure 8] This is a schematic diagram for constructing the plasmid pSWT004 of YB1-like Salmonella SWT005. [Figure 9] This is a validation diagram showing the identification of the enzyme digestion product of plasmid pSWT004 by agarose gel electrophoresis. [Figure 10] This is a verification diagram showing the identification of the cm-pepT-asd-sodA fragment by agarose gel electrophoresis of the PCR amplification product. [Figure 11] This is a validation diagram showing the identification of the PCR amplification product of Salmonella SWT005 by agarose gel electrophoresis. [Figure 12] This figure shows the results of an experiment verifying the oxygen adaptability of YB1-like Salmonella SWT005. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 13] This is a validation diagram showing the identification of the PCR amplification product of plasmid pSWT005 by agarose gel electrophoresis. [Figure 14] This is a verification diagram showing the identification of the PCR amplification product of the SWT006 strain by agarose gel electrophoresis. [Figure 15]This figure shows the results of oxygen adaptability testing of the SWT006 strain. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 16] This is a validation diagram of a bacterial strain library into which a hypoxia-specific gene expression cassette (alr is an essential gene) was inserted, as identified by agarose gel electrophoresis. (A) SWT1001, SWT1002, SWT1003, SWT1004, SWT1005, SWT1006, SWT1007, SWT1008 (B) SWT1009, SWT1010, SWT1011, SWT1012, SWT1013, SWT1014, SWT1015, SWT1016 (C) SWT1017, SWT1018, SWT1019, SWT1020, SWT1021, SWT1022, SWT1023, SWT1024 (D) SWT1025, SWT1026, SWT1027, SWT1028, SWT1029, SWT1030, SWT1031, SWT1032 (E) SWT1033, SWT1034, SWT1035, SWT1036, SWT1037, SWT1038, SWT1039, SWT1040 (F) SWT1041, SWT1042, SWT1043, SWT1044, SWT1045, SWT1046, SWT1047, SWT1048 (G)SWT1049, SWT1050, SWT1051, SWT1052, SWT1053, SWT1054, SWT1055, SWT1056 (H)SWT1057, SWT1058, SWT1059, SWT1060, SWT1061, SWT1062, SWT1063, SWT1064 (I)SWT1065, SWT1066, SWT1067, SWT1068, SWT1069, SWT1070, SWT1071, SWT1072 (J)SWT1073, SWT1074, SWT1075 [Figure 17] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1001. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 18]This figure shows the results of oxygen adaptability testing for the Salmonella strain SWT1002. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 19] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1004. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 20] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1005. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 21] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1009. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 22] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1010. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 23] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1012. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 24] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1013. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 25] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1021. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 26] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1025. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 27] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1033. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 28] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1035. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 29] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1036. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 30] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1038. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 31] This figure shows the results of oxygen adaptability testing for Salmonella strain SWT1063. (A) Cultured under anaerobic conditions on a D-alanine-free LB plate. (B) Cultured under aerobic conditions on a D-alanine-free LB plate. [Figure 32] This diagram shows the results of oxygen adaptability testing of various Salmonella strains when the oxygen concentration is 0.8% or less. (A) SWT1001 is cultured on an LB plate. (B) SWT1005 is cultured on an LB plate. (C) SWT1009 is cultured on an LB plate. (D) SWT1013 is cultured on an LB plate. [Figure 33]This is a distribution map of Salmonella tumors containing hypoxia-specific gene expression cassettes within hypoxic regions, as determined by immunohistochemical identification. (A) Tumor section stained with anti-Salmonella antibody: The brown areas indicated by arrows are areas where antibody staining is positive. (B) Tumor section stained with anti-HIF1α antibody: The brown areas indicated by arrows are areas where antibody staining is positive. [Figure 34] This diagram shows the invasion activity of strain SWT1001 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 35] Figure 34 is a supplement to the diagram showing the invasion effects of strain SWT1001 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 36]This diagram shows the invasion activity of strain SWT1002 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 37] Figure 36 is a supplement to the diagram showing the invasion effects of strain SWT1002 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 38] This diagram shows the invasion activity of strain SWT1004 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 39]Figure 38 is a supplement to the diagram showing the invasion effects of strain SWT1004 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 40] This diagram shows the invasion activity of strain SWT1005 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 41] Figure 40 is a supplement to the diagram showing the invasion effects of strain SWT1005 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage. [Figure 42]This diagram shows the invasion activity of strain SWT1009 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined image of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 43] Figure 42 is a supplement to the diagram showing the invasion effects of strain SWT1009 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 44] This diagram shows the invasion activity of strain SWT1010 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 45]Figure 44 is a supplement to the diagram showing the invasion effects of strain SWT1010 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 46] This diagram shows the invasion activity of strain SWT1012 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 47] Figure 46 is a supplement to the diagram showing the invasion effects of strain SWT1012 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 48]This diagram shows the invasion activity of strain SWT1013 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 49] Figure 48 is a supplement to the diagram showing the invasion effects of strain SWT1013 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 50] This diagram shows the invasion activity of strain SWT1021 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 51]Figure 50 is a supplement to the diagram showing the invasion effects of strain SWT1021 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 52] This diagram shows the invasion activity of strain SWT1025 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 53] Figure 52 is a supplement to the diagram showing the invasion effects of strain SWT1025 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 54]This diagram illustrates the invasion activity of strain SWT1033 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 55] Figure 54 is a supplement to the diagram showing the invasion effects of strain SWT1033 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 56] This diagram shows the invasion activity of strain SWT1035 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 57]Figure 56 is a supplement to the diagram showing the invasion effects of strain SWT1035 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 58] This diagram illustrates the invasion activity of strain SWT1036 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 59] Figure 58 is a supplement to the diagram showing the invasion effects of strain SWT1036 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 60]This diagram shows the invasion activity of strain SWT1038 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 61] Figure 60 is a supplement to the diagram showing the invasion effects of strain SWT1038 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 62] This diagram shows the invasion activity of strain SWT1063 against various cancer cell lines. BF shows bright-field images, FITC indicates the fluorescence signal of Salmonella after invasion, and Merge shows a combined view of BF and FITC. (A) Invasion activity against CT26 cell line (B) Invasion activity against EMT6 cell line (C) Invasion activity against MB49 cell line (D) Invasion activity against RM-1 cell line (E) Invasion activity against A549 cell line (F) Invasion activity against K7M2 cell line (G) Invasion activity against B16F10 cell line (H) Invasion activity against MFC cell line (I) Invasion activity against SCC7 cell line (J) Invasion activity against Hepa1-6 cell line (K) Invasion activity against Pan02 cell line (L) Invasion activity against ID8 cell line [Figure 63]Figure 62 is a supplement to the diagram showing the invasion effects of strain SWT1063 against various cancer cell lines. BF indicates bright-field imaging, the bright dots indicated by FITC are the fluorescence signals of Salmonella after invasion, and Merge is a combined diagram of BF and FITC. (M) Invasion effect against Renca cell lineage (N) Invasion effect against Neuro-2a cell lineage [Figure 64] This figure compares the toxic effects of bacterial strains SWT005, SWT1001, and SWT1002 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells. (A) This figure compares the toxic effects of bacterial strains SWT005 and SWT1001 on various cancer cell lines. (B) This figure compares the toxic effects of bacterial strains SWT005 and SWT1002 on various cancer cell lines. [Figure 65] This figure compares the toxic effects of strains SWT005, SWT1004, and SWT1005 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells. (A) This figure compares the toxic effects of strains SWT005 and SWT1004 on various cancer cell lines. (B) This figure compares the toxic effects of strains SWT005 and SWT1005 on various cancer cell lines. [Figure 66] This figure compares the toxic effects of bacterial strains SWT005, SWT1009, and SWT1010 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells. (A) This figure compares the toxic effects of bacterial strains SWT005 and SWT1009 on various cancer cell lines. (B) This figure compares the toxic effects of bacterial strains SWT005 and SWT1010 on various cancer cell lines. [Figure 67]This figure compares the toxic effects of comparative strains SWT005, SWT1012, and SWT1013 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells. (A) This figure compares the toxic effects of strains SWT005 and SWT1012 on various cancer cell lines. (B) This figure compares the toxic effects of strains SWT005 and SWT1013 on various cancer cell lines. [Figure 68] This figure compares the toxic effects of comparative strains SWT005, SWT1021, and SWT1025 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells. (A) This figure compares the toxic effects of strains SWT005 and SWT1021 on various cancer cell lines. (B) This figure compares the toxic effects of strains SWT005 and SWT1025 on various cancer cell lines. [Figure 69] This figure compares the toxic effects of bacterial strains SWT005, SWT1033, and SWT1035 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells. (A) This figure compares the toxic effects of bacterial strains SWT005 and SWT1033 on various cancer cell lines. (B) This figure compares the toxic effects of bacterial strains SWT005 and SWT1035 on various cancer cell lines. [Figure 70]This figure compares the toxic effects of bacterial strains SWT005, SWT1036, and SWT1038 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells. (A) This figure compares the toxic effects of bacterial strains SWT005 and SWT1036 on various cancer cell lines. (B) This figure compares the toxic effects of bacterial strains SWT005 and SWT1038 on various cancer cell lines. [Figure 71] This figure compares the toxic effects of bacterial strains SWT005 and SWT1063 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells. [Figure 72] This figure shows the targeting ability of the bacterial strain SWT005 in a mouse tumor model. The bacterial counts in the heart, liver, spleen, lung, kidney, and tumor on day 1 (D1), day 5 (D5), and day 11 (D11) after injecting SWT005 into mice via the tail vein. [Figure 73] This figure shows the targetability of strains SWT1001, SWT1002, SWT1004, and SWT1005 in a mouse tumor model. The bacterial counts are shown in the Heart, Liver, Spleen, Lung, Kidney, and Tumor on days 1 (D1), 5 (D5), and 11 (D11) after injection of the Salmonella strains into mice via the tail vein. Statistical analysis compares these results to strain SWT1005. (A) Strain SWT1001 (B) Strain SWT1002 (C) Strain SWT1004 (D) Strain SWT1005 [Figure 74]This figure shows the targetability of strains SWT1009, SWT1010, SWT1012, and SWT1013 in a mouse tumor model. The bacterial counts are shown in the Heart, Liver, Spleen, Lung, Kidney, and Tumor on days 1 (D1), 5 (D5), and 11 (D11) after injection of the Salmonella strains into mice via tail vein. Statistical analysis compares these results to strain SWT005. (A) Strain SWT1009 (B) Strain SWT1010 (C) Strain SWT1012 (D) Strain SWT1013 [Figure 75] This figure evaluates the targeting ability of strains SWT1021, SWT1025, SWT1033, and SWT1035 in a mouse tumor model. The figures show bacterial counts in the heart, liver, spleen, lung, kidney, and tumor on days 1 (D1), 5 (D5), and 11 (D11) after injection of the Salmonella strains into mice via the tail vein. Statistical analysis compares these results to strain SWT005. (A) Strain SWT1021 (B) Strain SWT1025 (C) Strain SWT1033 (D) Strain SWT1035 [Figure 76] This figure shows the targeting ability of strains SWT1036, SWT1038, and SWT1063 in a mouse tumor model. The bacterial counts are shown in the Heart, Liver, Spleen, Lung, Kidney, and Tumor on days 1 (D1), 5 (D5), and 11 (D11) after injection of the Salmonella strains into mice via the tail vein. Statistical analysis compares these results to strain SWT005. (A) Strain SWT1036 (B) Strain SWT1038 (C) Strain SWT1063 [Figure 77]This study compares the tumor-suppressing effects of strains SWT005, SWT1001, SWT1002, SWT1004, and SWT1005 in a mouse tumor model, with "Vehicle" representing the control group. (A) A comparison chart of the tumor-suppressing effects of strains SWT005 and SWT1001 in a mouse tumor model. (B) A comparison chart of the tumor-suppressing effects of strains SWT005 and SWT1002 in a mouse tumor model. (C) A comparison chart of the tumor-suppressing effects of strains SWT005 and SWT1004 in a mouse tumor model. (D) A comparison chart of the tumor-suppressing effects of strains SWT005 and SWT1005 in a mouse tumor model. [Figure 78] This study compares the tumor-suppressing effects of bacterial strains SWT005, SWT1009, SWT1010, SWT1012, and SWT1013 in a mouse tumor model, with "Vehicle" representing the control group. The graphs show: a comparison of the tumor-suppressing effects of strains SWT005 and SWT1009 in a mouse tumor model; a comparison of the tumor-suppressing effects of strains SWT005 and SWT1010 in a mouse tumor model; a comparison of the tumor-suppressing effects of strains SWT005 and SWT1012 in a mouse tumor model; and a comparison of the tumor-suppressing effects of strains SWT005 and SWT1013 in a mouse tumor model. [Figure 79] This study compares the tumor-suppressing effects of bacterial strains SWT005, SWT1021, SWT1025, SWT1033, and SWT1035 in a mouse tumor model, with "Vehicle" representing the control group. (A) A comparison chart of the tumor-suppressing effects of bacterial strains SWT005 and SWT1021 in a mouse tumor model. (B) A comparison chart of the tumor-suppressing effects of bacterial strains SWT005 and SWT1025 in a mouse tumor model. (C) A comparison chart of the tumor-suppressing effects of bacterial strains SWT005 and SWT1033 in a mouse tumor model. (D) A comparison chart of the tumor-suppressing effects of bacterial strains SWT005 and SWT1035 in a mouse tumor model. [Figure 80]This study compared the tumor-suppressing effects of bacterial strains SWT005, SWT1036, SWT1038, and SWT1063 in a mouse tumor model, with Vehicle being the control group. (A) A comparison chart of the tumor-suppressing effects of bacterial strains SWT005 and SWT1036 in a mouse tumor model. (B) A comparison chart of the tumor-suppressing effects of bacterial strains SWT005 and SWT1038 in a mouse tumor model. (C) A comparison chart of the tumor-suppressing effects of bacterial strains SWT005 and SWT1063 in a mouse tumor model. [Figure 81] This is a verification diagram showing the identification of the PCR amplification product of SWT007 by agarose gel electrophoresis. [Figure 82] This is a validation library diagram of strains identified by agarose gel electrophoresis of bacterial strains into which a hypoxia-specific gene expression cassette (dadX is an essential gene) was inserted. SWT2001, SWT2002, SWT2004, SWT2005, SWT2009, SWT2010, SWT2012, SWT2013, SWT2033, SWT2035, SWT2036, SWT2038, SWT2021, SWT2025, SWT2063 [Figure 83] This diagram shows the oxygen adaptability of strains SWT2001, SWT2002, SWT2004, and SWT2005. (B) SWT2001 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (C) SWT2004 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (D) SWT2005 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. [Figure 84]These are diagrams illustrating the oxygen adaptability of strains SWT2009, SWT2010, SWT2012, and SWT2013. (B) SWT2009 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (C) SWT2010 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (D) SWT2013 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. [Figure 85] This diagram shows the oxygen adaptability of strains SWT2033, SWT2035, SWT2036, and SWT2038. (B) SWT2033 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (C) SWT2036 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (D) SWT2038 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. [Figure 86] This diagram illustrates the oxygen adaptability of strains SWT2021, SWT2025, and SWT2063. (B) SWT2021 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (C) SWT2063 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. [Figure 87]This diagram shows the oxygen adaptability of strains SWT1065, SWT1066, SWT1068, and SWT1069. (B) SWT1065 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (C) SWT1068 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (D) SWT1069 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. [Figure 88] This diagram shows the oxygen adaptability of strains SWT1041, SWT1042, SWT1044, and SWT1045. (B) SWT1041 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (C) SWT1044 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (D) SWT1045 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. [Figure 89] This diagram shows the oxygen adaptability of strains SWT1008, SWT1016, and SWT1024. (B) SWT1008 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (C) SWT1016 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. [Figure 90]This study verifies the oxygen adaptability of strains SWT1073, SWT1074, and SWT1075. (A) SWT1073 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (B) SWT1074 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. (C) SWT1075 was cultured under anaerobic conditions in the upper section and under aerobic conditions in the lower section of a D-alanine-free LB plate. [Modes for carrying out the invention]

[0144] The present application will be described in more detail below with reference to the drawings and embodiments. These descriptions will make the features and advantages of the present application clearer and more apparent. Example 1: Construction of a hypoxia-specific gene expression cassette with the alur gene as a survival-essential gene.

[0145] In this example, the following were adopted as forward hypoxia promoters: Salmonella yhbU (yhbU-S) (SEQ ID No. 1), Escherichia coli ynfK (ynfK-E) (SEQ ID No. 2), Salmonella tdcA (tdcA-S) (SEQ ID No. 3), Escherichia coli tdcA (tdcA-E) (SEQ ID No. 4), Escherichia coli yecH (yecH-E) (SEQ ID No. 5), Salmonella ynfK (ynfK-S) (SEQ ID No. 6), Escherichia coli cydA (cydA-E) (SEQ ID No. 7), Escherichia coli focA (focA-E) (SEQ ID No. 8), and Escherichia coli yhbU (yhbU-E) (SEQ ID No. 48), respectively. The Salmonella alr gene (SEQ ID No. 48) was adopted as the essential gene for survival. Using ID No. 17), Salmonella cyoA (cyoA-S) (SEQ ID No. 9), Salmonella phoH (phoH-S) (SEQ ID No. 10), Salmonella lldP ​​(lldP-S) (SEQ ID No. 11), Salmonella argT (argT-S) (SEQ ID No. 12), Salmonella ydcI (ydcI-S) (SEQ ID No. 13), Escherichia coli phoH (phoH-E) (SEQ ID No. 14), Escherichia coli lldP ​​(lldP-E) (SEQ ID No. 15), Escherichia coli ydcI (ydcI-E) (SEQ ID No. 16), and Escherichia coli mqo (SEQ ID No. 62) were used as reverse hyperoxygen promoters to construct hypoxia-specific gene expression cassettes.

[0146] Table 1 shows the combinations of hypoxia-specific gene expression cassettes.

[0147] [Table 1] (1) Construction of a forward hypoxia promoter cloning library

[0148] 1. A single clone of wild-type Salmonella typhi (strain SWT001, purchased from CICC China Industrial Microbial Species Collection Center) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) was selected, inoculated into 5 ml of LB liquid medium, and incubated for 16 hours in a constant temperature shaker at 37°C and 220 rpm. OD 600 The value is between 2 and 3.

[0149] 2. Using a pipette tip, 2 μl of the corresponding bacterial strain culture was aspirated, mixed with the corresponding primer and a high-fidelity PCR amplification enzyme (purchased from TAKARA Corporation), and placed in a PCR amplification instrument. The amplification program consisted of 2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 60°C, 60 seconds at 72°C, 30 cycles, 10 minutes at 72°C, and 5 minutes at 4°C.

[0150] 3. The amplified PCR product was purified and recovered using a DNA gel recovery system.

[0151] 4. The recovered product was mixed with an endonuclease (purchased from NEB) enzyme digestion system and incubated at 37°C for 1 hour.

[0152] 5. The enzyme digestion products were purified and recovered using a DNA gel recovery system. The primers and endonucleases used for each forward hypoxia promoter are as follows:

[0153] 1. yhbU-S (SEQ ID No. 1): Strain SWT001 was amplified using primers SWTO19 and 20, and the product was double-digested with NotI and HindIII before being recovered. 2. ynfK-E (SEQ ID No. 2): Escherichia coli strain DH10B was amplified using primers SWTO21 and 22, and the product was double-digested with NotI and HindIII before being recovered. 3. tdcA-S (SEQ ID No. 3): Strain SWT001 was amplified using primers SWTO23 and 24, and the product was double-digested with NotI and HindIII before being recovered. 4. tdcA-E (SEQ ID No. 4): Escherichia coli strain DH10B was amplified using primers SWTO25 and 26, and the product was double-digested with NotI and HindIII before being recovered. 5. yecH-E (SEQ ID No. 1): Escherichia coli strain DH10B was amplified using primers SWTO27 and 28, and the residue was double-digested with NotI and HindIII before being collected. 6. ynfK-S (SEQ ID No. 6): S strain WT001 was amplified using primers SWTO29 and 30, and the product was double-digested with NotI and HindIII before being recovered. 7. cydA-E (SEQ ID No. 7): Escherichia coli strain DH10B was amplified using primers SWTO31 and 32, and the product was double-digested with NotI and HindIII before being recovered. 8. focA-E (SEQ ID No. 8): Escherichia coli strain DH10B was amplified using primers SWTO33 and 34, and the product was double-digested with NotI and HindIII before being recovered. 9. yhbU-E (SEQ ID No. 48): Escherichia coli strain DH10B was amplified using primers SWTO82 and 83, and the product was double-digested with NotI and HindIII before being recovered.

[0154] The results of verifying the forward-directed hypoxic promoter clone library by agarose gel electrophoresis are shown in Figures 1(A) and 1(B). (ii) Construction of a library of genes essential for survival

[0155] 1. A single clone of wild-type Salmonella typhi (strain SWT001, purchased from CICC China Industrial Microbial Species Collection Center) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) was selected, inoculated into 5 ml of LB liquid medium, and incubated for 16 hours in a constant temperature shaker at 37°C and 220 rpm.

[0156] 2. Using a pipette tip, 2 μl of the corresponding bacterial strain culture was aspirated, mixed with the corresponding primer and high-fidelity PCR amplification enzyme system, and placed into the PCR amplification instrument. The amplification program consisted of 2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 60°C, 60 seconds at 72°C, 30 cycles, 10 minutes at 72°C, and 5 minutes at 4°C.

[0157] 3. The amplified PCR product was purified and recovered using a DNA gel recovery system.

[0158] 4. The recovered product was mixed with an enzyme digestion system and incubated at 37°C for 1 hour.

[0159] 5. The enzyme digestion products were purified and recovered using a DNA gel recovery system.

[0160] The essential genes for survival obtained from different sources are as follows: 1. Salmonella alr gene (SEQ ID No. 17): Strain SWT001 was amplified using primers SWTO35 and SWTO36, and the product was double-digested with HindIII and XhoI before being recovered. 2. Escherichia coli alr gene (SEQ ID No. 21): Strain DH10B was amplified using primers SWTO37 and 38, and the product was double-digested with HindIII and XhoI before being recovered. 3. Salmonella dadX gene (SEQ ID No. 19): Strain SWT001 was amplified using primers SWTO39 and 40, and the product was double-digested with HindIII and XhoI before being recovered. 4. Escherichia coli dadX gene (SEQ ID No. 23): Strain DH10B was amplified using primers SWTO41 and 42, and the product was double-digested with HindIII and XhoI before being recovered.

[0161] Figure 2 shows the results of validation of the survival essential gene library by agarose gel electrophoresis. (3) Construction of a reverse-direction hyperoxygen promoter library

[0162] 1. A single clone of wild-type Salmonella typhi (strain SWT001, purchased from CICC China Industrial Microbial Species Collection Center) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) was selected, inoculated into 5 ml of LB liquid medium, and incubated for 16 hours in a constant temperature shaker at 37°C and 220 rpm.

[0163] 2. Using a pipette tip, 2 μl of the corresponding bacterial strain culture was aspirated, mixed with the corresponding primer and high-fidelity PCR amplification enzyme system, and placed into the PCR amplification instrument. The amplification program consisted of 2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 60°C, 60 seconds at 72°C, 30 cycles, 10 minutes at 72°C, and 5 minutes at 4°C.

[0164] 3. The amplified PCR product was purified and recovered using a DNA gel recovery system.

[0165] 4. The recovered product was mixed with an enzyme digestion system and incubated at 37°C for 1 hour.

[0166] 5. The enzyme digestion products were purified and recovered using a DNA gel recovery system.

[0167] The primers and endonucleases used for each of the reverse hyperoxygen promoters are as follows: 1. cyoA-S (SEQ ID No. 9): Strain SWT001 was amplified using primers SWTO43 and 44, and the product was double-digested with XhoI and PstI before being recovered. 2. phoH-S (SEQ ID No. 10): Strain SWT001 was amplified using primers SWTO45 and 46, and the product was double-digested with XhoI and PstI before being recovered. 3. lldP-S (SEQ ID No. 11): Strain SWT001 was amplified using primers SWTO47 and 48, and the product was double-digested with XhoI and PstI before being recovered. 4. argT-S (SEQ ID No. 12): Strain SWT001 was amplified using primers SWTO49 and 50, and the product was double-digested with XhoI and PstI before being recovered. 5. ydcI-S (SEQ ID No. 13): Strain SWT001 was amplified using primers SWTO51 and 52, and the product was double-digested with XhoI and PstI before being recovered. 6. phoH-E (SEQ ID No. 14): Strain DH10B was amplified using primers SWTO53 and 54, and the product was double-digested with XhoI and PstI before being recovered. 7. lldP-E (SEQ ID No. 15): Strain DH10B was amplified using primers SWTO55 and 56, and the product was double-digested with XhoI and PstI before being recovered. 8. ydcI-E (SEQ ID No. 16): Strain DH10B was amplified using primers SWTO57 and 58, and the product was double-digested with XhoI and PstI before being recovered. 9. mqo (SEQ ID No. 62): Strain DH10B was amplified using primers SWTO7 and 8, and the product was double-digested with XhoI and PstI before being recovered. The results of verifying the reverse hyperoxygen promoter clone library by agarose gel electrophoresis are shown in Figures 3(A) and 3(B). (iv) Construction of a library of combinations of forward hypoxia promoters, survival essential genes, and reverse hyperxia promoters.

[0168] As shown in Figure 4, the hypoxia-specific gene expression cassette is composed of a forward hypoxia promoter, essential survival genes, and a reverse hyperxia promoter in that order.

[0169] As shown in Table 1, clonal libraries were constructed by combining sequences from forward hypoxia promoter libraries digested with NotI and HindIII, the Salmonella alr gene (SEQ ID No. 17), a survival-essential gene, digested with HindIII and XhoI, and reverse hyperxia promoter libraries digested with XhoI and PstI. These libraries were then ligated into plasmid pSWT003 vector (vector skeleton, pBlueScript SK(+), purchased from Seibutsu-fusha) digested with SpeI and PstI, and plasmid pSWT007 vector (containing bilateral symphatic loxp sequences and a DNA fragment of the chloramphenicol resistance gene (SEQ ID No. 61)) digested with SpeI and PstI.

[0170] The above-mentioned Salmonella SWT001 or Escherichia coli DH10B were amplified using primers, and the products recovered by enzymatic digestion were combined and ligated by an enzymatic ligation reaction. These products were then spread onto LB plates containing 25 μg / ml chloramphenicol to obtain the corresponding plasmids shown in Table 1.

[0171] The construction of each strain of this invention is as follows. 1. Wild-type Salmonella typhi (SWT001)

[0172] Wild-type Salmonella typhi (SWT001) was purchased from the CICC China Industrial Microbial Species Collection Center. 2. Construction of Salmonella (SWT002) containing temperature-inducible lambda-RED recombinase and loxp-CRE enzyme system.

[0173] The lambda-RED recombinant system is widely used for homologous recombination in Gram-negative bacteria. In this invention, this system consists of plasmid pSWT001. As shown in Figure 5, plasmid pSWT001 contains a lambda-RED recombinase module (SEQ ID No. 60) (functionally similar to the plasmid vector psim6 from Seibutsu-Keishakusha) and a loxp-Cre recombinase module (SEQ ID No. 60) (functionally similar to the plasmid vector 705-Cre from Gene Bridges). The lambda-RED recombinase module consists of three recombinases, EXO, BET, and GAM, and these recombinases are controlled by the temperature regulator CI857. Therefore, expression does not occur under culture conditions of 32°C, but only when the temperature exceeds 37°C. Under temperature-induced conditions, recombinase EXO cleaves the 5' end of double-stranded linear DNA, forming single-stranded DNA with a protruding 3' end. This single-stranded DNA binds to the BET protein and is protected from degradation by other nucleases. GAM plays a role in inhibiting the action of endogenous bacterial nucleases. Homologous recombination homologous arms are approximately 35-50 bp long and are added to both sides of the DNA fragment that requires recombination by PCR. The advantage of this technique is that it precisely targets the desired region on the bacterial chromosome without causing additional mutations. The recombinant double-stranded DNA fragment contains homologous arm sequences at the recombination site on both outermost layers, and inside are bilateral sympathetic loxp sequences totaling 34 bp. Inside the sympathetic double loxp region is the chloramphenicol resistance gene used for screening recombinant bacteria. If recombination is successful, the CRE enzyme system (similarly regulated by the CI857 temperature regulator) mounted on the pSWT001 plasmid specifically recognizes the loxp sequence and cleaves the central sequence of the sympathetic loxp, leaving a single loxp sequence, thereby removing the chloramphenicol resistance gene. By repeating this process, continuous knockout and knock-in of genes becomes possible.

[0174] The specific steps are as follows: (1) Streak the strain SWT001 on an LB plate, and perform static culture overnight in a constant temperature incubator at 37°C. (2) Select a single clone, inoculate it into 5 mL of LB liquid medium, place the medium in a constant temperature shaker at 37°C and 220 rpm, and perform shake culture for 16 hours. (3) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100, continue culturing for 2 to 3 hours until the bacterial density reaches an OD 600 of 0.3, then place the culture on ice and let it stand for 1 hour. (4) Wash the bacterial cells 3 times with sterile purified water. (5) Mix the collected bacterial cells with 10 ng of plasmid pSWT001, and perform electroporation at a voltage of 1.8 kV. (6) Spread the electroporated bacterial cells on an LB plate containing 100 μg / mL ampicillin sodium, culture overnight in a constant temperature incubator at 32°C until a single clonal colony grows, and name this strain SWT002. 3. Construction of attenuated Salmonella (SWT003) with aroA gene knockout

[0175] (1) Select a single clone of SWT002, inoculate it into 5 mL of LB liquid medium containing 100 μg / mL ampicillin sodium, place the medium in a constant temperature shaker at 32°C and 220 rpm, and culture for 16 hours. (2) Inoculate the culture into fresh LB liquid medium containing 100 μg / mL ampicillin sodium at a ratio of 1:100, continue culturing for 2 to 3 hours until the bacterial density reaches an OD 600 of 0.3, place the culture flask in a 42°C water bath for 15 minutes of shake culture, then place it on ice and let it stand for 1 hour. (3) Wash the bacterial cells 3 times with sterile purified water. (4) Prepare PCR products of SWTO1 and SWTO2. Mix primers SWTO1 and SWTO2 with plasmid pSWT002 (which contains loxp sequences in the same direction on both sides and a chloramphenicol resistance gene in the middle) and a high-fidelity PCR amplification enzyme system, then load the mixture into a PCR amplification instrument. The amplification program was: 2 min at 95°C, 30 sec at 95°C, 30 sec at 60°C, 60 sec at 72°C, 30 cycles, 10 min at 72°C, and 5 min at 4°C. The amplified PCR products were subjected to agarose gel electrophoresis to verify the PCR amplification products of SWTO1 and SWTO2 of the aroA gene knockout fragment, and the verification results are shown in Figure 6A. The products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were measured by Nanodrop. (5) The recovered bacterial cells were mixed with 100 ng of the PCR products of SWTO1 and SWTO2, and electroporation was performed at a voltage of 1.8 kV. (6) The electroporated bacterial cells were spread on a plate containing 25 μg / ml chloramphenicol, and cultured overnight in a constant temperature incubator at 32°C until single clonal colonies grew. (7) Positive clones were identified by colony PCR. Insertion of the chloramphenicol resistance gene was confirmed using SWTO4 and SWTO6, as well as SWTO3 and SWTO5. The PCR amplification product of the target fragment of strain SWT003 was verified by agarose gel electrophoresis, and the verification result is shown in Figure 6B. (8) The positive single clone was inoculated into 5 mL of LB medium, and cultured for 16 hours in a constant temperature shaker at 37°C and 220 rpm. CRE enzyme was allowed to act to remove the chloramphenicol resistance gene. 4. Construction of attenuated bacterium (SWT004) with alr gene knockout

[0176] (1) A single clone of SWT003 was selected, inoculated into 5 mL of LB liquid medium, and placed in a constant temperature shaker at 32°C and 220 rpm to culture for 16 hours. (2) The culture was inoculated into fresh LB liquid medium at a ratio of 1:100, and the culture was continued until the bacterial density OD 600 reached 0.3, after which the culture flask was placed in a 42°C water bath, cultured with shaking for 15 minutes, then placed on ice and allowed to stand for 1 hour. (3) The bacterial cells were washed 3 times with sterile purified water. (4) PCR products of SWTO70 and SWTO71 were prepared. Primers SWTO70 and SWTO71 were mixed with plasmid pSWT002 and a high-fidelity PCR amplification enzyme system, and then placed in a PCR amplification instrument. The amplification program consisted of 2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 60°C, 60 seconds at 72°C, 30 cycles, 10 minutes at 72°C, and 5 minutes at 4°C. The amplified PCR products were purified and recovered using a DNA gel recovery system. The concentration and purity of the recovered PCR products were measured using nanodrops. (5) The recovered bacterial cells were mixed with 100 ng of the PCR products of SWTO70 and SWTO71, and electroporation was performed at a voltage of 1.8 kV. The PCR amplification products of the arr gene knockout fragments SWTO70 and SWTO71 were verified by agarose gel electrophoresis, and the verification results are shown in Figure 7A. (6) The electroporated bacterial cells were spread onto plates containing 25 μg / ml chloramphenicol and incubated overnight in a 32°C constant temperature incubator until single clonal colonies grew. (7) Positive clones were identified by colony PCR. Insertion of the chloramphenicol resistance gene was confirmed using SWTO72 and SWTO5, and SWTO6 and SWTO73. The PCR amplification product of the target fragment of strain SWT004 was validated by agarose gel electrophoresis, and the validation results are shown in Figure 7B (the primers used were SWTO72, SWTO5, SWTO6, and SWTO73). (8) Positive single clones were inoculated into 5 ml of LB medium and cultured for 16 hours in a constant temperature shaker at 37°C and 220 rpm, and the chloramphenicol resistance gene was removed by treatment with CRE enzyme. 5. Construction of YB1-like Salmonella (SWT005)

[0177] In order to study a more preferred combination of oxygen control promoters, the present invention constructed a strain of Salmonella YB1 described in Chinese patent CN104471057B and compared it with the strain constructed in the present invention as a prior art strain.

[0178] YB1-like Salmonella strains include those with aroA gene deficiency and oxygen regulatory systems constructed from the forward hypoxia promoter pepT, the essential gene asd, and the reverse hyperxia promoter sodA. (1) Construction of plasmid pSWT004

[0179] ○1 As shown in Figure 8, the pSWT004 plasmid was constructed containing the chloramphenicol resistance gene cm (SEQ ID No. 43), pepT promoter (SEQ ID No. 44), asd gene (SEQ ID No. 45), and reverse sodA promoter (SEQ ID No. 47), each possessing a bilateral loxp sequence.

[0180] ○2 Plasmid pSWT002 was digested with NotI, and 1130 bp fragments were recovered. Strain SWT001 was amplified using primers SWTO11 and SWTO12, and the product was recovered by double digestion with NotI and HindIII. Strain SWT001 was amplified using primers SWTO13 and SWTO14, and the product was recovered by double digestion with XhoI and HindIII. XhoI and PstI nicks were generated and recovered by direct annealing with primers SWTO15 and SWTO16.

[0181] ○3 Plasmid pSWT003 (plasmid backbone) was double digested with NotI and PstI, and the five fragments were ligated using T4 DNA ligase. These fragments were then spread onto a dual-resistance LB plate for ampicillin and chloramphenicol, and a single clone was screened and named pSWT004 (a YB1-like construct plasmid containing cm-pepT-asd-sodA).

[0182] ○4 Plasmid pSWT004 was extracted, 100 ng was mixed with SacI and KpnI endonuclease systems, incubated at 37°C for 1 hour, and identified by agarose gel electrophoresis as shown in Figure 9. (2) Construction of recombinant YB1-like Salmonella SWT005 (YB1-like)

[0183] Plasmid pSWT004 was amplified and recovered using primers SWTO17 and SWTO18, and this fragment was recombined into SWT003 to construct YB1-like Salmonella SWT005.

[0184] ○1 A single clone of SWT003 was selected, inoculated into 5 ml of LB liquid medium, and incubated for 16 hours in a constant temperature shaker at 32°C and 220 rpm.

[0185] ○2 Inoculate the culture into fresh LB liquid medium at a ratio of 1:100, and when the bacterial density reaches OD 600 Continue culturing for 2-3 hours until the value reaches 0.3, then place the culture flask in a 42°C water bath and shake for 15 minutes, after which place it on ice and let it stand for 1 hour.

[0186] ○3 The bacterial cells were washed three times with sterile purified water and prepared for use.

[0187] ○4 pSWT004 was amplified using primers SWTO17 and SWTO18, purified, and recovered. The recovered PCR products were examined by agarose gel electrophoresis to determine the PCR amplification products of the cm-pepT-asd-sodA fragments SWTO17 and SWTO18, and the results are shown in Figure 10. The concentration and purity of the DNA were measured using nanodrops.

[0188] ○5 The recovered bacterial cells were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV.

[0189] ○6 The bacterial cells subjected to electroporation were spread on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml DAP (diaminopimelic acid), and cultured overnight in a constant temperature incubator at 32°C until single clonal colonies grew.

[0190] ○7 Positive clones were identified by colony PCR. The insertion of the chloramphenicol resistance gene was confirmed using SWTO93 and SWTO5, as well as SWTO94 and SWTO95. Figure 11 shows the verification results of PCR amplification products of Salmonella SWT005 by agarose gel electrophoresis (SWTO93 and SWTO5, SWTO94 and SWTO95). (3) Verification of oxygen adaptability of YB1-like Salmonella SWT005

[0191] ○1 A single clone of YB1-like Salmonella SWT005 was selected, inoculated into 5 ml of LB liquid medium containing 100 μg / ml DAP (diaminopimelic acid), and cultured for 16 hours in a constant temperature shaker at 37°C and 200 rpm.

[0192] ○2 After completion of culture, the strain was diluted 10-fold, and the absorbance (OD value) at 600 nm was measured.

[0193] ○3 The volume of bacterial solution (μL) for 1 OD of bacteria was calculated by the following formula, and deionized water was added to make up to 1 ml. Volume of bacterial solution for 1 OD of bacteria = 1 / (absorbance×10)×1000

[0194] ○4 10 μl of the above bacterial solution was spread onto 2 DAP-free LB plates, marked as "1". This procedure was repeated 3 times, and the repeats were marked as "a", "b", and "c".

[0195] ○5 After 10-fold serial dilution, another 10 μl of the dilution was spread onto the above medium, marked as "2".

[0196] ○6 This 10-fold serial dilution was continued until the dilution mark reached "8".

[0197] ○7 One plate was placed in an anaerobic tank at 37°C. For the procedure, an anaerobic gas generating bag (manufacturer: Mitsubishi Nippon, part number: D-119) was placed in a 7.0L sealed culture tank (manufacturer: Mitsubishi Nippon, part number: D-112), and cultured in an anaerobic environment (oxygen concentration less than 0.01%) for 24 hours. The other plate was cultured in an atmospheric environment at 37°C (oxygen concentration 21%) for 24 hours.

[0198] Figure 12 shows the results of the oxygen adaptability verification test for YB1-like Salmonella SWT005.

[0199] From the oxygen adaptability verification data of YB1-like Salmonella SWT005, it was found that YB1-like Salmonella SWT005 can grow normally on DAP-free LB plates under anaerobic conditions (see Figure 12(A)), but under aerobic conditions (atmospheric conditions), it cannot grow at a concentration of 1 (10 -2 OD), concentration 2(10 -3 OD), concentration 3(10 -4 OD), concentration 4(10 -5 In both OD (Od) conditions, clear growth of the bacterial strain was observed (see Figure 12(B)).

[0200] Therefore, it was found that YB1-like Salmonella SWT005 does not have a tight control over the growth of the strain in an aerobic environment. This is consistent with previous reports on YB1 and further suggests that there is a specific defect in the oxygen regulation system of YB1 Salmonella SWT005.

[0201] Therefore, the present invention proposes an improved form that achieves stricter control of bacteria under aerobic conditions and enhances the tendency of bacteria to target hypoxic regions of tumors by substituting essential gene fragments on the one hand and pre- and post-promoter fragments on the other hand. 6. Verification of essential gene substitutions and construction of the SWT006 strain.

[0202] Based on experimental data from YB1-like Salmonella SWT005, this invention suggests that the selection of the essential gene asd in YB1-like Salmonella SWT005 (pepT-asd-sodA strain) is not optimal.

[0203] Therefore, in this invention, the essential gene asd in the oxygen-regulating expression cassette of YB1-like Salmonella SWT005 was replaced with the alr gene to construct a new hypoxia-specific regulatory expression cassette (pepT-alr-sodA), and its oxygen-regulating function was verified.

[0204] Since only double mutations in the alr and dadX genes can induce a lethal trait, it is necessary to first construct the SWT004 strain (a strain in which the aroA gene and the alr gene are knocked out), and then incorporate a hypoxia-specific regulatory expression cassette (pepT-alr-sodA) into the dadX gene region of the SWT004 strain (disrupting its function) to construct the SWT006 strain. (1) Construction of plasmid pSWT005

[0205] Based on SWT004, a hypoxia-specific gene expression cassette containing a pepT promoter, the essential gene alr, and a reverse sodA promoter was incorporated into the SWT004 dadX gene site. The construction process is as follows:

[0206] ○1 Plasmid pSWT004 (a YB1-like plasmid containing cm-pepT-asd-sodA) was digested with HindIII and XhoI, the asd fragment was removed, and the remaining fragment was recovered.

[0207] ○2 Strain SWT001 was amplified using primers SWTO35 and SWTO36 to obtain amplified alr gene products of Salmonella, and the products were double digested with HindIII and XhoI and recovered.

[0208] ○3 The above fragments were ligated using T4 DNA ligase.

[0209] ○4 DH10B competent cells were transformed, plated onto LB plates containing both ampicillin and chloramphenicol, and inverted for 16 hours at 37°C.

[0210] ○5 A single clone was selected and PCR identification was performed using primers SWTO11 and SWTO36. The PCR amplification products of SWTO11 and SWTO36 were verified by agarose gel electrophoresis, and the verification results are shown in Figure 13.

[0211] ○6 Bacteria were cultured by shaking, and plasmids were extracted to obtain plasmid pSWT005 (cm-pepT-alr-sodA element). (2) Hypoxia-specific gene expression cassette pepT-alr-sodA strain (SWT006, aroA - &alr - Construction of (&pepT-alr-sodA)

[0212] ○1 A single clone of attenuated SWT004 with the alr gene knocked out was selected, inoculated into 5 ml of LB liquid medium, and incubated for 16 hours in a constant temperature shaker at 32°C and 220 rpm.

[0213] ○2 Inoculate the culture into fresh LB liquid medium at a ratio of 1:100, and when the bacterial density reaches OD 600 Continue culturing for 2-3 hours until the value reaches 0.3, then place the culture flask in a 42°C water bath and shake for 15 minutes, after which place it on ice and let it stand for 1 hour.

[0214] ○3 The bacterial cells were washed three times with sterile purified water and prepared for use.

[0215] ○4 pSWT005(cm-pepT-alr-sodA) was amplified using primers SWTO89 and SWTO90, purified, and recovered. The concentration and purity of the recovered PCR product DNA were measured using nanodrops.

[0216] ○5 The recovered bacterial cells were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV.

[0217] ○6 The electroporated bacterial cells were spread onto plates containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and incubated overnight in a 32°C constant temperature incubator until single clonal colonies grew.

[0218] ○7 Positive clones were identified by colony PCR. Insertion of the chloramphenicol resistance gene was confirmed in SWTO87 and SWTO76, and in SWTO59 and SWTO77. The PCR amplification products of Salmonella SWT006 were verified by agarose gel electrophoresis, and the verification results are shown in Figure 14. (3) Verification of oxygen adaptability of strain SWT006

[0219] ○1 A single clone of strain SWT006 was inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine and incubated for 16 hours in a constant temperature shaker at 37°C and 200 rpm.

[0220] ○2 After the culture was completed, the bacterial strain was diluted 10-fold and the absorbance (OD value) at 600 nm was measured.

[0221] ○The volume of bacterial suspension for 1 OD bacteria was calculated using a cubic equation, and deionized water was added up to 1 ml. The liquid volume of a 1OD bacterium = 1 / (absorbance × 10) × 1000

[0222] ○4 10 μl of the above bacterial suspension was spread onto two D-alanine-free LB plates and labeled "1". This was repeated three times and labeled "a", "b", and "c".

[0223] ○5 After serial dilution 10 times, another 10 μl of the diluted solution was spread onto the culture medium and labeled "2".

[0224] ○6 Continue this 10-fold serial dilution until the result reaches "8".

[0225] One plate was cultured at 37°C in an anaerobic environment (oxygen concentration less than 0.01%), and the other plate was cultured at 37°C in an atmospheric environment (oxygen concentration 21%).

[0226] The results of the oxygen adaptability verification test of the SWT006 strain are shown in Figure 15(A). The SWT006 strain can grow normally under anaerobic environments.

[0227] As can be seen from the oxygen adaptability verification results of the SWT006 strain, compared with the experimental results of the oxygen adaptability verification of SWT005 YB1-like, the selection of the essential gene alr is better controlled under aerobic conditions than the asd gene of SWT005 YB1-like. As shown in Figure 15(B), concentration 1 (10 -2 OD), concentration 2 (10 -3 OD), and concentration 3 (10 -4 OD), obvious growth of the strain was only observed.

[0228] In order to further optimize the present invention and achieve stricter oxygen control, the forward hypoxia-specific promoter and the reverse high-oxygen promoter of the hypoxia-specific gene expression cassette are further adjusted. Example 2: Integration of a hypoxia-specific gene expression cassette into the dadX gene site on the chromosome of SWT004 strain (disruption of its function).

[0229] (1) Construction of alr-deficient attenuated bacteria containing a hypoxia-specific gene expression cassette

[0230] 1. A single clone of the attenuated bacterium SWT004 with the alr gene knocked out was selected, inoculated into 5 mL of fresh LB liquid medium, and cultured for 16 hours in a constant temperature shaker at 32°C and 220 rpm.

[0231] 2. The culture was inoculated into fresh LB liquid medium at a ratio of 1:100, and the culture was continued for 2 to 3 hours until the bacterial density reached OD 600 = 0.3. The culture flask was placed in a 42°C water bath, cultured with shaking for 15 minutes, then placed on ice and allowed to stand for 1 hour.

[0232] 3. The bacterial cells were washed three times with sterile purified water.

[0233] 4. PCR products were prepared to construct a strain containing a hypoxia-specific expression cassette. The primers SWTO78 and SWTO79 were mixed with the hypoxia-specific gene expression cassette plasmid and a high-fidelity PCR amplification enzyme system, and then placed in a PCR amplification instrument. The amplification program consisted of 2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 60°C, 120 seconds at 72°C (30 cycles), 10 minutes at 72°C, and 5 minutes at 4°C. 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 measured using nanodrops.

[0234] 5. The recovered bacterial cells were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV.

[0235] 6. The electroporated bacterial cells were spread onto plates containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and incubated overnight in a 32°C constant temperature incubator until single clonal colonies grew.

[0236] 7. Single clonal colonies were inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, incubated at 37°C and 220 rpm in a constant temperature shaker for 16 hours, and treated with CRE enzyme to remove the chloramphenicol resistance gene.

[0237] 8. Using the corresponding primers, the clones were identified by colony PCR, and the identification results are shown in Figure 16.

[0238] Table 2 shows information on the corresponding bacterial strains, and Table 3 shows the correspondence between the bacterial strains and identification primers.

[0239] [Table 2(1)] [Table 2(2)] [Table 2(3)] [Table 2(4)] [Table 2(5)] [Table 2(6)] [Table 2(7)]

[0240] [Table 3(1)] [Table 3(2)] (ii) Verification of oxygen adaptability of a Salmonella strain library including a hypoxia-specific gene expression cassette.

[0241] 1. A single clone of a strain containing a hypoxia-specific expression cassette was selected, inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and incubated for 16 hours in a constant temperature shaker at 37°C and 200 rpm.

[0242] 2. After the culture was complete, the bacterial strain was diluted 10-fold and the absorbance (OD value) at 600 nm was measured.

[0243] 3. The volume of bacterial suspension for 1 OD was calculated using the following formula, and deionized water was added up to 1 ml. The liquid volume of a 1OD bacterium = 1 / (absorbance × 10) × 1000

[0244] 4. 10 μl of the above bacterial suspension was inoculated onto two D-alanine-free LB plates and labeled "1". This was repeated three times and labeled "a", "b", and "c".

[0245] After serial dilution 5.10 times, another 10 μl of the diluted solution was inoculated into the culture medium and labeled "2".

[0246] 6. Continue this 10-fold serial dilution until the result reaches "8".

[0247] 7. One plate was cultured at 37°C under anaerobic conditions, and the other plate was cultured at 37°C under atmospheric conditions (oxygen concentration 21%).

[0248] Figures 17-31 show the results of oxygen adaptability testing of strains containing hypoxia-specific expression cassettes.

[0249] Based on data from the YB1-like strain (SWT005) and the essential gene replacement strain (SWT006), the present invention designed a new forward hypoxia promoter, an essential gene, and a reverse hyperxia promoter, forming a new hypoxia-specific gene expression cassette. As a result, Salmonella strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063, all containing the hypoxia-specific gene expression cassette, showed superior performance in mutation evaluations compared to the YB1-like strain (SWT005) and the essential gene replacement strain (SWT006). (iii) Simulation of oxygen concentration for a bacterial strain library including a hypoxia-specific gene expression cassette.

[0250] By using an anaerobic gas generating bag to consume oxygen in a sealed culture tank and measuring the oxygen concentration in the tank with an oxygen meter, it is possible to stabilize the oxygen concentration in the sealed culture tank within a specific range after using the anaerobic gas generating bag for a certain period. The specific method is as follows. An anaerobic gas generating bag (brand: Mitsubishi Nippon, part number: D-119) was placed in a 7.0L sealed culture tank (brand: Mitsubishi Nippon, part number: D-112), and an oxygen meter (brand: Umejo Denka, part number: OX-100A) was also placed inside.

[0251] Using the above combination of anaerobic gas generating bags, we verified the growth of genetically modified bacterial strains at oxygen concentrations of 0.8% or less. 1. Verification of the adaptability of a strain library containing a hypoxia-specific gene expression cassette at oxygen concentrations of 0.8% or less.

[0252] Since the pathological hypoxic region of a tumor is an area where the oxygen concentration is less than 1%, the present invention aims to demonstrate that Salmonella bacteria modified with a hypoxia-specific gene expression cassette can grow normally even when the oxygen concentration is less than 1% (or close to 1%).

[0253] Oxygen concentration was controlled by an anaerobic gas generating bag. One hour after oxygen consumption by the anaerobic gas generating bag, the oxygen meter reading indicated an oxygen concentration of 0.8-1%. Therefore, in this experiment, we simulated a hypoxic environment within the tumor and verified the growth of Salmonella bacteria on a culture dish one hour after oxygen consumption by the anaerobic gas generating bag, as well as simulating the growth of Salmonella strains containing hypoxia-specific gene expression cassettes in a hypoxic environment within the tumor.

[0254] The procedure for the TCL experiment is as follows:

[0255] (1) A single clone of a strain containing a hypoxia-specific expression cassette was selected, inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and incubated for 16 hours in a constant temperature shaker at 37°C and 200 rpm. (2) After the culture was completed, the bacterial strain was diluted 10-fold and the absorbance (OD value) at 600 nm was measured. (3) The volume of bacterial suspension for 1 OD bacteria was calculated using the following formula, and deionized water was added up to 1 ml. The liquid volume of a 1OD bacterium = 1 / (absorbance × 10) × 1000 (4) 10 μl of the above bacterial suspension was inoculated into an LB plate containing D-alanine and cultured, and labeled "1". This was repeated three times and labeled "a", "b", and "c". (5) Simultaneously, 10 μl of the above bacterial suspension was inoculated into another LB plate that did not contain D-alanine and cultured, and labeled "1". This was repeated three times and labeled "a", "b", and "c". (6) After 10-fold serial dilution, another 10 μl of the diluted solution was inoculated into the culture medium described above and labeled "2". (7) Continue this 10-fold serial dilution until the result is "8". (8) Two plates were cultured at 37°C in a 0.8% oxygen environment. As shown in Figure 32, the present invention compared the growth of various bacterial strains (Figure 32(A) SWT1001, Figure 32(B) SWT1005, Figure 32(C) SWT1009, Figure 32(D) SWT1013) on LB plates without D-alanine supplementation, and determined the growth status of each strain at oxygen concentrations of 0.8% or less. Since the corresponding Salmonella strains need to rely on their own oxygen control system for normal growth in a medium without D-alanine supplementation, this result supports the idea that all strains containing hypoxia-specific expression cassettes that can grow normally in an anaerobic environment can also grow normally at oxygen concentrations of 0.8% or less. 2. Immunohistochemical staining of hypoxic areas of tumors with a bacterial strain (SWT1005) containing a hypoxia-specific gene expression cassette.

[0256] To demonstrate the aggregation of Salmonella containing hypoxia-specific gene expression cassettes in hypoxic regions of tumors, mouse tumor models injected with Salmonella via the tail vein were sectioned, stained for Salmonella and hypoxia-inducible factor HIF-1α antibodies, and the distribution of Salmonella in the tumor models was observed. (1) Establishment of tumor models

[0257] BALB / c mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., weighing approximately 18g, raised in an SPF environment) were given 1 x 10⁶ doses. 6A mouse colon cancer subcutaneous tumor model was constructed by subcutaneously inoculating CT26 mouse colon cells. 10-12 days after inoculation, the tumor volume was approximately 100 mm². 3 The experiment was conducted when the following conditions were met. The mice were divided into two groups of five: the PBS group and the SWT1005 group. The tumor-bearing mice were given 1 × 10⁶ of 125 μl of solution. 7 CFU bacteria were inoculated via the tail vein. (2) Preparation of bacteria

[0258] Taking strain SWT1005 as an example, it was streaked onto an LB plate with added D-alanine and incubated overnight in a 37°C constant temperature incubator. A single clone of strain SWT1005 was selected, inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and incubated for 16 hours in a 37°C, 220 rpm constant temperature shaker. The absorbance (OD value) of the strain at 600 nm was measured, and the culture with an OD value of 1 was weighed, washed three times with PBS, and a PBS bacterial suspension was prepared. (3) Preparation of tumor sections

[0259] Three days after administration, the animals were killed, dissected, the tumors removed, and immersed in formalin fixative. They were dehydrated by gradient and embedded in paraffin. After complete cooling and solidification, sections were prepared and dewaxed. The sections were immersed in sodium citrate antigen retrieval solution, boiled for 10 minutes to retrieve the antigen, and then washed three times with PBS. The antigen-retrieved sections were immersed in 3% hydrogen peroxide solution for 10 minutes and washed three times with PBS. The sections after catalase removal were immersed in 2% BSA-PBS solution, blocked at room temperature for 1 hour, and then washed three times with PBS. Anti-salmonella (purchased from Abcam, catalog no. ab35156) and anti-HIF1α (purchased from Abcam, catalog no. ab51608) primary antibody incubation solutions were dropped onto different blocked tissue sections, incubated overnight at 4°C, and then washed three times with PBS. Secondary antibody incubation solution was added to the tissue sections and incubated for 25 minutes, then washed three times with PBS. DAB staining solution was added to the tissue sections and counterstained with hematoxylin for 3 minutes. After gradient dehydration of the tissue sections, they were mounted with mounting medium, observed under a microscope, and photographed, as shown in Figure 33.

[0260] The results, after antibody staining and comparison, showed that the brown (dark) areas were antibody-positive, indicating that Salmonella bacteria containing hypoxia-specific gene expression cassettes were accumulating in the hypoxic areas of the tumor. (iv) Evaluation of the cell invasion effect of bacterial strains containing hypoxia-specific gene expression cassettes

[0261] Salmonella cells modified with a hypoxia-specific gene expression cassette were transformed with pSWT006 (an EGFP-heavy expression plasmid controlled by a prokaryotic promoter). Invasion of tumor cells by Salmonella was determined by observing the location of fluorescence and comparing cell distribution.

[0262] The invasive effects of bacterial strains containing hypoxia-specific gene expression cassettes on cells of various cancers, including breast cancer (EMT6 cell lineage), osteosarcoma (K7M2 cell lineage), liver cancer (Hepa1-6 cell lineage), lung cancer (A549 cell lineage), melanoma (B16F10 cell lineage), kidney cancer (Renca cell lineage), gastric cancer (MFC cell lineage), pancreatic cancer (Pan02 cell lineage), prostate cancer (RM-1 cell lineage), colon cancer (CT26 cell lineage), ovarian cancer (ID8 cell lineage), neuroblastoma (Neuro-2a cell lineage), squamous cell carcinoma (SCC7 cell lineage), and bladder cancer (MB49 cell lineage), were evaluated. 1. Evaluation of the invasion activity of strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063, modified with hypoxia-specific gene expression cassettes, against cancer cells.

[0263] (1) By electroporating a bacterial strain modified with a hypoxia-specific gene expression cassette onto plasmid pSWT006 and expressing the EGFP protein, we facilitated the observation of cell invasion. (2) A strain modified with a hypoxia-specific gene expression cassette carrying pSWT006 was streaked onto an LB plate supplemented with D-alanine, and cultured statically overnight in a 37°C constant temperature incubator. (3) A single clone of the modified strain carrying pSWT006 was inoculated into 5 mL of fresh LB liquid medium containing 100 µg / mL D-alanine, placed in a constant temperature shaker at 37°C and 220 rpm, and cultured for 16 hours. (4) A 1 OD culture was weighed and washed three times with PBS. (5) Into each well of a 6-well plate for cell culture, 2.5×10 6 corresponding cancer cells were added, and cultured overnight in a complete medium (DMEM basal medium + 10% fetal bovine serum + double antibody) in a 37°C, 5% CO2 incubator.

[0264] After counting cells from two wells, strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063 modified with a hypoxia-specific gene expression cassette carrying pSWT006 were added at a multiplicity of infection of 200:1 based on cell count. The cells were placed in an anaerobic tank with an oxygen concentration of 0.8% or less, and co-cultured for 2 hours to allow the bacteria to invade the cells. Thereafter, the cells were gently washed with an equal volume of PBS, and the supernatant was removed by aspiration. DMEM medium containing 100 µg / mL gentamicin was added, and the cells were further cultured for 24 hours in the anaerobic tank with an oxygen concentration of 0.8% or less, and photographs were taken with a fluorescence microscope. The invasive effects of the strains modified with the hypoxia-specific gene expression cassette against various cancer cell lines are shown in Figures 34 to 63.

[0265] The above results show that Salmonella carrying a hypoxia-specific gene expression cassette has strong invasiveness against various cancer cell lines. The intracellular growth and distribution of various Salmonella strains carrying the hypoxia-specific gene expression cassette have been clearly observed, demonstrating that Salmonella has a universal invasive ability against cancer cells. 2. Killing effect of strains containing hypoxia-specific gene expression cassettes on cancer cells under hypoxic conditions.

[0266] The killing effects of strain SWT005 (YB1-like) and Salmonella strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063, which contain the hypoxia-specific gene expression cassette of the present invention, against various cancer cells were verified by co-culturing them with cancer cells under in vitro hypoxia conditions.

[0267] (1) Strain SWT005 was streaked onto LB plates with added DAP. Modified strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063 were streaked onto LB plates with added D-alanine and incubated overnight in a 37°C constant temperature incubator. (2) A single clone of strain SWT005 was selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml DAP. Single clones of the modified strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038 and SWT1063 were each selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and incubated in a constant temperature shaker at 37°C and 220 rpm for 16 hours. (3) The absorbance of the bacterial strain at 600 nm was measured, and the culture with an OD value of 1 was weighed, washed twice with PBS, washed once with the corresponding cell medium, and then the bacterial cells were resuspended in the cell medium (infection ratio 200:1). (4) Each of the above bacteria was co-cultured with EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7, and MB49 cells at an oxygen concentration of 0.8% or less for 2 hours. The cells were then washed three times with PBS containing gentamicin, the culture medium was changed, and the culture was continued in an anaerobic tank at an oxygen concentration of 0.8% or less for 24 hours. (5) The CCK8 reagent was mixed with the cell suspension described above and incubated for 1 hour. (6) Absorbance was measured using a microplate reader (detection wavelength 450 nm) and the cytotoxicity rate was calculated.

[0268] Figures 64-71 show the toxic effects of each strain SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063, each containing a hypoxia-specific gene expression cassette, on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7, and MB49 cells compared to bacterium SWT005.

[0269] The experimental results were analyzed using t-tests, and p<0.05(*), p<0.01(**), and p<=0.001(***) were shown to be statistically significant.

[0270] The results above demonstrate that all Salmonella strains modified with hypoxia-specific gene expression cassettes exhibit significant killing activity against various cancer cells, and this activity is more potent than that of strain SWT005 (YB1-like), indicating that Salmonella modified with hypoxia-specific gene expression cassettes has a universal killing effect against cancer cells. 3. Evaluation of the targetability of bacterial strains containing hypoxia-specific gene expression cassettes in mouse tumor models.

[0271] To investigate the tumor targeting potential of bacterial strains containing hypoxia-specific gene expression cassettes in mouse tumor models, we established an EMT6 tumor mouse model.

[0272] BALB / c mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., weighing approximately 18g, raised in a Specified Pathogen Free (SPF) environment) were given 1 x 10⁶ doses. 6 A mouse mammary gland subcutaneous tumor model was created by subcutaneously inoculating individual EMT6 cancer cells. 14-18 days after inoculation, the tumor volume was approximately 500 mm³. 3 The experiment was conducted when the condition was reached.

[0273] The tumor-bearing mice were randomly divided into 17 groups of 6 mice each: PBS (Vehicle) group, SWT005 group, SWT1001 group, SWT1002 group, SWT1004 group, SWT1005 group, SWT1009 group, SWT1010 group, SWT1012 group, SWT1013 group, SWT1021 group, SWT1025 group, SWT1033 group, SWT1035 group, SWT1036 group, SWT1038 group, and SWT1063 group. The remaining 16 groups of tumor-bearing mice were given 1 × 10⁶ oz solution in a volume of 125 μl. 7 CFU bacteria were administered intravenously via the tail vein. Mice were sacrificed and dissected on day 1 (D1), day 5 (D5), and day 11 (D11) after administration. The quantitative distribution of these strains in normal tissues and organs of tumor-bearing mice, including the heart, liver, spleen, lungs, kidneys, and tumors, was analyzed. The results of evaluating the targetability of each Salmonella strain, including hypoxia-specific gene expression cassettes, in the mouse tumor model were compared with YB1-like strains, as shown in Figures 72-76.

[0274] The results above indicate that Salmonella strains containing hypoxia-specific gene expression cassettes, compared to YB1-like strains, were less distributed at all time points in the heart, liver, spleen, lungs, and kidneys of the mouse model, but accumulated rapidly within tumors and were significantly more abundant than YB1-like strains. (There are no columns indicating complete elimination of Salmonella.)

[0275] The experimental results were analyzed using t-tests, and p<0.05(*), p<0.01(**), and p<=0.001(***) were shown to be statistically significant. 4. Evaluation of tumor suppression in mouse tumor models of bacterial strains containing hypoxia-specific gene expression cassettes.

[0276] To verify the inhibitory effect of strains containing a hypoxia-specific gene expression cassette on mouse tumor growth, the present invention designed inhibitory tests in a mouse EMT6 tumor model for the YB1-like strain SWT005, as well as Salmonella strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063 containing a hypoxia-specific gene expression cassette.

[0277] Creation of an EMT6 tumor mouse model: BALB / c mice (purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., approximately 18g body weight, raised in a Specified Pathogen Free (SPF) environment) were given 1 × 10⁶ samples. 6 A mouse mammary gland subcutaneous tumor model was created by subcutaneously inoculating individual EMT6 cancer cells. 14-18 days after inoculation, the tumor volume was approximately 500 mm³. 3 The experiment was conducted when the condition was reached.

[0278] (1) Strain SWT005 was streaked onto LB plates to which DAP had been added, and the constructed Salmonella strain was streaked onto LB plates to which D-alanine had been added, and the cells were incubated statically overnight in a 37°C constant temperature incubator. (2) A single clone of strain SWT005 was selected and inoculated into 5 ml of LB liquid medium to which DAP had been added. Single clones of the constructed Salmonella strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063 were selected and inoculated into 5 ml of LB liquid medium to which D-alanine had been added, and incubated in a constant temperature shaker at 37°C and 220 rpm for 16 hours. (3) The absorbance of the bacterial strain at 600 nm was measured, and the culture with an OD value of 1 was weighed and washed three times with PBS. (4) 1 × 10⁻¹⁰ 7 CFU bacteria were injected into EMT6 model mice via the tail vein. (5) The day of administration was designated as day 0, and the length and width of the mouse tumors were measured on days 1, 3, 5, 7, 9, and 11 after administration, respectively, and the tumor volume was calculated using the following formula. Mouse tumor volume = length × width 2 ×0.52

[0279] Figures 77-80 show the tumor-suppressing effects of various Salmonella strains, including those containing hypoxia-specific gene expression cassettes, in mouse tumor models compared to the YB1-like strain SWT005.

[0280] The experimental results were analyzed using t-tests, and p<0.05(*), p<0.01(**), and p<=0.001(***) were shown to be statistically significant.

[0281] Conclusion: Salmonella strains containing hypoxia-specific gene expression cassettes significantly inhibited tumor growth and were superior to the YB1-like strain SWT005. Example 3: Construction of a hypoxia-specific gene expression cassette using the dadX gene as a survival-essential gene.

[0282] As alanine racemase, bacteria such as Salmonella and Escherichia coli possess two isozyme genes in their genomes: the alfr gene and the dadX gene. To verify their effects, the oxygen-regulating effect of the hypoxia-specific gene expression cassette, which had been verified above, was investigated by replacing the survival-essential gene from the alfr gene to the corresponding homologous gene dadX. (1) Integration of the hypoxia-specific gene expression cassette into the alr gene site of the SWT007 strain chromosome

[0283] 1. Construction of a weakened bacterium with the dadX gene knocked out (SWT007)

[0284] (1) A single clone of SWT003 was selected, inoculated into 5 ml of LB liquid medium, and incubated in a constant temperature shaker at 32°C and 220 rpm for 16 hours. (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and check the bacterial density 600 Continue culturing for 2-3 hours until the value reaches 0.3, then place the culture flask in a 42°C water bath and shake for 15 minutes, after which place it on ice and let it stand for 1 hour. (3) The bacterial cells were washed three times with sterile purified water. (4) PCR products of SWTO78 and SWTO79 were prepared. Primers SWTO78 and SWTO79 were mixed with plasmid pSWT002 (plasmid as described above) and a high-fidelity PCR amplification enzyme system, and then placed in a PCR amplification instrument. The amplification program consisted of 2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 60°C, 60 seconds at 72°C, 30 cycles, 10 minutes at 72°C, and 5 minutes at 4°C. 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 measured using nanodrops. (5) The recovered bacterial cells were mixed with 100 ng of the PCR products of SWTO78 and SWTO79, and electroporation was performed at a voltage of 1.8 kV. (6) The electroporated bacterial cells were spread onto plates containing 25 μg / ml chloramphenicol and incubated overnight in a 32°C constant temperature incubator until single clonal colonies grew. (7) Positive clones were identified by colony PCR. As shown in Figure 81, insertion of the chloramphenicol resistance gene was confirmed using SWTO59, SWTO5, SWTO6, and SWTO87. (8) Positive single clones were inoculated into 5 ml of LB medium and cultured for 16 hours in a constant temperature shaker at 37°C and 220 rpm, and the chloramphenicol resistance gene was removed by treatment with CRE enzyme. 2. Construction of a plasmid containing a hypoxia-specific gene expression cassette and requiring the Salmonella dadX gene as an essential gene.

[0285] (1) Using a pipette tip, a single clone of the Salmonella strain SWT001 was selected, mixed with primers SWTO39 and SWTO40, and a high-fidelity PCR amplification enzyme, and placed in a PCR amplification instrument. The amplification program consisted of 2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 60°C, 60 seconds at 72°C, 30 cycles, 10 minutes at 72°C, and 5 minutes at 4°C. (2) The amplified PCR product (Salmonella dadX gene (SEQ ID No. 19)) was purified and recovered using a DNA gel recovery system, as shown in Figure 2. (3) The recovered products were mixed with HindIII and XhoI enzyme digestion systems and incubated at 37°C for 1 hour. The digested products were purified and recovered using a DNA gel recovery system. (4) 5 μg each of plasmids pOL1001, pOL1002, pOL1004, pOL1005, pOL1009, pOL1010, pOL1012, pOL1013, pOL1021, pOL1025, pOL1033, pOL1035, pOL1036, pOL1038, and pOL1063 was taken, mixed with HindIII and XhoI enzyme digestion systems respectively, incubated at 37°C for 1 hour, the vector fragments were recovered, mixed with the fragments from step (3), and ligated under the action of T4 DNA ligase. (5) DH10B competent cells were transformed, plated with chloramphenicol and ampicillin, and a single clone was selected. (6) The vectors that were successfully constructed are pOL2001, pOL2002, pOL2004, pOL2005, pOL2009, pOL2010, pOL2012, pOL2013, pOL2021, pOL2025, pOL2033, pOL2035, pOL2036, pOL2038, and pOL2063, as shown in Table 4.

[0286] [Table 4]

[0287] The survival-essential gene in Table 4 is the Salmonella dadX gene.

[0288] 3. Construction of a deficient, attenuated bacterium containing a hypoxia-specific gene expression cassette, with the Salmonella dadX gene as an essential survival gene.

[0289] (1) A single clone of the attenuated bacterium SWT007 with the dadX gene knocked out was selected, inoculated into 5 ml of LB liquid medium, and cultured for 16 hours in a constant temperature shaker at 32°C and 220 rpm. (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and check the bacterial density 600 Continue culturing for 2-3 hours until the value reaches 0.3, then place the culture flask in a 42°C water bath and shake for 15 minutes, after which place it on ice and let it stand for 1 hour. (3) The bacterial cells were washed three times with sterile purified water. (4) PCR products were prepared to construct bacterial strains containing hypoxia-specific expression cassettes. Primers SWTO85 and SWTO86 were mixed with the hypoxia-specific gene expression cassette plasmid (see Table 4) and a high-fidelity PCR amplification enzyme system, and placed in a PCR amplification instrument. The amplification program consisted of 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, and 5 minutes at 4°C. The amplified PCR products were purified and recovered using a DNA gel recovery system. The concentration and purity of the recovered PCR products were measured using nanodrops. (5) The recovered bacterial cells were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV. (6) The electroporated bacterial cells were spread onto plates containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and incubated overnight in a 32°C constant temperature incubation chamber until a single clone grew. (7) Positive clones were inoculated into 5 ml of LB medium and cultured for 16 hours in a constant temperature shaker at 37°C and 220 rpm, and the chloramphenicol resistance gene was removed by treatment with CRE enzyme. (8) Clones were identified by colony PCR using the corresponding primers. The identification results are shown in Figure 82. The correspondence between the bacterial strains and the identification primers is shown in Table 5.

[0290] [Table 5] (ii) Oxygen adaptability verification test of a library of Salmonella strains containing a hypoxia-specific gene expression cassette and in which Salmonella dadX is a survival-essential gene (dadX gene knocked out and expression cassette incorporated into Salmonella arl gene).

[0291] (1) A single clone of the bacterial strain was selected, inoculated into 5 mL of LB liquid medium supplemented with D-alanine, and incubated for 16 hours in a constant temperature shaker at 37°C and 200 rpm. (2) After the culture was completed, the bacterial strain was diluted 10-fold and the absorbance (OD value) at 600 nm was measured. (3) The volume of bacterial suspension for 1 OD bacteria was calculated using the following formula, and deionized water was added to make a total volume of 1 mL. The liquid volume of a 1OD bacterium = 1 / (absorbance × 10) × 1000 (4) 10 μl of the above bacterial suspension was inoculated onto two D-alanine-free LB plates and labeled "1". This was repeated three times and labeled "a", "b", and "c". (5) After 10-fold serial dilution, another 10 μl of the diluted solution was inoculated into the culture medium described above and labeled "2". (6) Continue the 10-fold serial dilution in this manner until the result is "6". (7) One plate was cultured in an anaerobic environment at 37°C, and the other plate was cultured in an atmospheric environment (oxygen concentration 21%) at 37°C.

[0292] Figures 83-86 show the results of oxygen adaptability testing of strains containing hypoxia-specific expression cassettes.

[0293] The results above indicate that the essential gene for the hypoxia-specific gene expression cassette can be either the arl gene or the dadX gene, and that both exert similar oxygen-regulating effects. Example 4: Study on the replacement of source bacteria for the construction of a forward hypoxia promoter.

[0294] This embodiment was designed to replenish and expand the same forward hypoxia promoter from different source bacteria. (1) Forward hypoxia promoter yhbU

[0295] In the present invention, the forward hypoxia promoter used in strains SWT1001, SWT1002, SWT1004, and SWT1005 is Salmonella yhbU. The forward hypoxia promoter used in strains SWT1065, SWT1066, SWT1068, and SWT1069 is Escherichia coli yhbU.

[0296] In the case of Gram-negative facultative anaerobic bacteria, the promoter yhbU contains a conserved FNR binding site, and all of these FNR binding sites are characterized by having the sequence "CTGCCNNNNATCAA", where N represents one of the bases A, T, C, or G. The promoter yhbU is the forward hypoxic promoter "TTGATNNNNATCAA", and is characterized by the fact that any of the bases in the TTGAT and ATCAA sequences of the conserved binding site are substitutable, but the total number of substitutions is 3 or less, and it is not possible to substitute three consecutive bases.

[0297] The present invention validates the yhbU promoters of Salmonella and Escherichia coli, and the results show that all yhbUs that fit this conserved site possess the corresponding oxygen control function and can be used as forward hypoxia promoters for the hypoxia-specific gene expression cassette described in the present invention.

[0298] Therefore, the yhbU promoter used in the hypoxia-specific gene expression cassette designed in the present invention can be derived from Salmonella (SEQ ID No. 1), Escherichia coli (SEQ ID No. 48), Shigella (SEQ ID No. 49), Yersinia (SEQ ID No. 50), Enterobacter cloacae (SEQ ID No. 51), Chronobacter (SEQ ID No. 52), Klebsiella (SEQ ID No. 53), Pantoea (SEQ ID No. 54), Serratia (SEQ ID No. 55), or Simwellia (SEQ ID No. 56).

[0299] Analysis of the yhbU promoter sequences of each of the above source bacteria revealed that although the promoter sequences differed significantly, their FNR binding sites were completely identical. [ka] It became clear that this was the case. Study on verifying oxygen adaptability of strains containing hypoxia-specific gene expression cassettes for *E. coli* yhbU.

[0300] (1) Single clones were selected from strains SWT1065, SWT1066, SWT1068, and SWT1069, inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and incubated in a constant temperature shaker at 37°C and 200 rpm for 16 hours. (2) After the culture was completed, the bacterial strain was diluted 10-fold and the absorbance (OD value) at 600 nm was measured. (3) The volume of bacterial suspension for 1 OD bacteria was calculated using the following formula, and deionized water was added until the volume reached 1 ml. The liquid volume of a 1OD bacterium = 1 / (absorbance × 10) × 1000 (4) 10 μl of the above bacterial suspension was inoculated onto two D-alanine-free LB plates and labeled "1". This was repeated three times and labeled "a", "b", and "c". (5) After 10-fold serial dilution, another 10 μl of the diluted solution was inoculated into the culture medium described above and labeled "2". (6) Continue the 10-fold serial dilution in this manner until the result is "6". (7) One plate was cultured at 37°C under anaerobic conditions, and the other plate was cultured at 37°C under atmospheric conditions (oxygen concentration 21%).

[0301] Figure 87 shows the results of oxygen adaptability testing for Salmonella strains SWT1065, SWT1066, SWT1068, and SWT1069.

[0302] These results demonstrate that a similar oxygen control effect can be achieved by replacing the forward hypoxia promoter from Salmonella-derived to E. coli-derived. (2) Forward hypoxic promoter ynfK

[0303] The forward hypoxia promoter used in strains SWT1009, SWT1010, SWT1012, and SWT1013 is E. coli ynfK.

[0304] The forward hypoxia promoter used in strains SWT1041, SWT1042, SWT1044, and SWT1045 is Salmonella ynfK.

[0305] In the case of Gram-negative facultative anaerobic bacteria, all promoter ynfKs contain a conserved FNR binding site, which is characterized by having the sequence "TTGCGNNNNCTCAA", where N represents one of the bases A, T, C, or G. The promoter ynfK is the forward hypoxic promoter "TTGATNNNNATCAA", which is characterized by the fact that any base in the TTGAT and ATCAA sequences of the conserved binding site is substitutable, but the total number of substitutions is 3 or less, and it is not possible to substitute three consecutive bases.

[0306] This invention validated Salmonella, Escherichia coli, and ynfK promoters. The results showed that all ynfKs compatible with this conserved site possessed the corresponding oxygen control function and could be used as forward hypoxia promoters for the hypoxia-specific gene expression cassette described in this invention.

[0307] Therefore, the ynfK promoter used in the hypoxia-specific gene expression cassette designed in the present invention can be derived from Salmonella (SEQ ID No. 6), Escherichia coli (SEQ ID No. 2), Serratia (SEQ ID No. 57), Shigella (SEQ ID No. 58), and Enterobacter ludwigia (SEQ ID No. 59). Study on verifying the oxygen adaptability of strains containing a hypoxia-specific gene expression cassette for the Salmonella ynfK promoter.

[0308] (1) Single clones were selected from strains SWT1041, SWT1042, SWT1044, and SWT1045, inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and incubated for 16 hours in a constant temperature shaker at 37°C and 200 rpm. (2) After the culture was completed, the bacterial strain was diluted 10-fold and the absorbance (OD value) at 600 nm was measured. (3) The volume of bacterial suspension for 1 OD bacteria was calculated using the following formula, and deionized water was added to make a total volume of 1 ml. The liquid volume of a 1OD bacterium = 1 / (absorbance × 10) × 1000 (4) 10 μl of the above bacterial suspension was inoculated onto two D-alanine-free LB plates and labeled "1". This was repeated three times and labeled "a", "b", and "c". (5) After 10-fold serial dilution, another 10 μl of the diluted solution was inoculated into the culture medium described above and labeled "2". (6) Continue the 10-fold serial dilution in this manner until the result is "6". (7) One plate was cultured at 37°C under anaerobic conditions, and the other plate was cultured at 37°C under atmospheric conditions (oxygen concentration 21%).

[0309] Figure 88 shows the results of oxygen adaptability testing for Salmonella strains SWT1041, SWT1042, SWT1044, and SWT1045. These results indicate that a similar oxygen control effect can be achieved by replacing the forward hypoxia promoter from E. coli to that derived from Salmonella.

[0310] The results above demonstrate, for example, that two promoters, yhbU and ynfK, possess highly conserved FNR binding sites in different facultative anaerobic bacteria. When these two promoters, derived from Escherichia coli and Salmonella, were incorporated into a hypoxia-specific gene expression cassette and compared, both were found to exhibit consistent oxygen control functions. From this, it can be concluded that the hypoxia-specific gene expression cassette described in the present invention can be constructed using promoters of the same gene possessing conserved FNR binding sites in different facultative anaerobic bacteria, and that similar effects can be achieved in both cases. Example 5: Study on the replacement of source bacteria for the construction of a reverse high-oxygen promoter.

[0311] In this invention, this embodiment is designed to replenish and expand the same reverse-direction hyperoxygen promoter from different source bacteria. (1) Reverse direction hyperoxygen promoter ydcI

[0312] In the present invention, the reverse hyperoxygen promoter used in strains SWT1005, SWT1013, and SWT1021 is the Salmonella ydcI promoter.

[0313] The reverse hyperoxygen promoter used in strains SWT1008, SWT1016, and SWT1024 is the E. coli ydcI promoter.

[0314] In the case of Gram-negative facultative anaerobic bacteria, the promoter ydcI contains a conserved FNR binding site, and all of these FNR binding sites have the characteristic sequence "CTGCCNNNNATCAA", where N represents one of the bases A, T, C, or G. The promoter ydcI is the reverse hyperoxygen promoter "TTGATNNNNATCAA", and it matches the characteristic that any of the bases in the conserved binding sites TTGAT and ATCAA sequences are substitutable, but the total number of substitutions is 3 or less, and it is not possible to substitute three consecutive bases. Furthermore, the ArcA binding site of the reverse hyperoxygen promoter matches the pattern GTTAATTA, and it also matches the characteristic that any of the bases are substitutable, but the total number of substitutions is 2 or less.

[0315] This invention validated the ydcI promoters of Salmonella and Escherichia coli. The results showed that all ydcI molecules matching this conserved site possessed the corresponding oxygen control function and could be used as reverse hyperoxygen promoters for the hypoxia-specific gene expression cassette described in this invention.

[0316] Therefore, the ydcI promoter used in the hypoxia-specific gene expression cassette designed in the present invention can be derived from Salmonella (SEQ ID No. 13) and Escherichia coli (SEQ ID No. 16). Research on verifying oxygen adaptability of strains containing a hypoxia-specific gene expression cassette including the E. coli ydcI promoter.

[0317] (1) Single clones of strains SWT1008, SWT1016, and SWT1024 were selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and incubated in a constant temperature shaker at 37°C and 200 rpm for 16 hours. (2) After the culture was completed, the bacterial strain was diluted 10-fold and the absorbance (OD value) at 600 nm was measured. (3) The volume of bacterial suspension for 1 OD bacteria was calculated using the following formula, and deionized water was added to make a total volume of 1 ml. The liquid volume of a 1OD bacterium = 1 / (absorbance × 10) × 1000 (4) 10 μl of the above bacterial suspension was inoculated onto two D-alanine-free LB plates and labeled "1". This was repeated three times and labeled "a", "b", and "c". (5) After 10-fold serial dilution, another 10 μl of the diluted solution was inoculated into the culture medium described above and labeled "2". (6) Continue the 10-fold serial dilution in this manner until the result is "6". (7) One plate was cultured at 37°C under anaerobic conditions, and the other plate was cultured at 37°C under atmospheric conditions (oxygen concentration 21%).

[0318] Figure 89 shows the results of oxygen adaptability testing for Salmonella strains SWT1008, SWT1016, and SWT1024.

[0319] These results demonstrate that a similar oxygen control effect can be obtained even when the forward hyperoxygen promoter is replaced from Salmonella-derived to E. coli-derived. (ii) Reverse hyperoxygen promoter mqo containing two ArcA binding sites

[0320] In this invention, the reverse hyperoxygen promoter used in strains SWT1004, SWT1012, and SWT1036 is the Salmonella argT promoter (SEQ ID No. 12).

[0321] The reverse hyperoxygen promoter used in strains SWT1073, SWT1074, and SWT1075 is the E. coli MQO promoter (SEQ ID No. 62).

[0322] Both the argT promoter (SEQ ID No. 12) and the mgo promoter (SEQ ID No. 62) contain a conserved double ArcA binding site and match the GTTAATTA pattern of the ArcA binding site of the reverse hyperoxygen promoter, where any of the bases are substituted, but the total number of substitutions is 2 or less. The present invention verified the argT promoter (SEQ ID No. 12) and the mgo promoter (SEQ ID No. 62) and demonstrated that all promoters containing a conserved double ArcA binding site have the corresponding oxygen control function and can function as reverse hyperoxygen promoters used in the hypoxia-specific gene expression cassette described in the present invention.

[0323] Therefore, the reverse hyperoxygen promoter used in the hypoxia-specific gene expression cassette designed in the present invention can be derived from an argT promoter (SEQ ID No. 12) and an mqo promoter (SEQ ID No. 62) that include a double ArcA binding site. Research on verifying oxygen adaptability of bacterial strains containing hypoxia-specific gene expression cassettes, including the MQO promoter.

[0324] (1) Single clones of strains SWT1073, SWT1074, and SWT1075 were selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and incubated in a constant temperature shaker at 37°C and 200 rpm for 16 hours. (2) After the culture was completed, the bacterial strain was diluted 10-fold and the absorbance (OD value) at 600 nm was measured. (3) The volume of bacterial suspension for 1 OD bacteria was calculated using the following formula, and deionized water was added to make a total volume of 1 ml. The liquid volume of a 1OD bacterium = 1 / (absorbance × 10) × 1000 (4) 10 μl of the above bacterial suspension was inoculated onto two D-alanine-free LB plates and labeled "1". This was repeated three times and labeled "a", "b", and "c". (5) After 10-fold serial dilution, another 10 μl of the diluted solution was inoculated into the culture medium described above and labeled "2". (6) Continue the 10-fold serial dilution in this manner until the result is "6". (7) One plate was cultured at 37°C under anaerobic conditions, and the other plate was cultured at 37°C under atmospheric conditions (oxygen concentration 21%).

[0325] Figure 90 shows the results of oxygen adaptability testing for Salmonella strains SWT1073, SWT1074, and SWT1075.

[0326] The results showed that a similar oxygen control effect could be obtained even when the forward hyperoxygen promoter was replaced from Salmonella-derived to E. coli-derived.

[0327] Table 6 shows the specific sequences of the forward hypoxia promoter, survival essential gene, and reverse hyperxia promoter of the present invention.

[0328] [Table 6(1)] [Table 6(2)] [Table 6(3)] [Table 6(4)] [Table 6(5)] [Table 6(6)] [Table 6(7)] [Table 6(8)] [Table 6(9)] [Table 6(10)] [Table 6(11)] [Table 6(12)] [Table 6(13)] [Table 6(14)] [Table 6(15)] [Table 6(16)] [Table 6(17)] [Table 6(18)] [Table 6(19)] [Table 6(20)] [Table 6(21)] [Table 6(22)]

[0329] Table 7 shows the specific sequences of all the primers used in this invention.

[0330] [Table 7(1)] [Table 7(2)] [Table 7(3)] [Table 7(4)]

[0331] The tool plasmid of the present invention is shown in Table 8.

[0332] [Table 8]

[0333] Although this application has been described with reference to the above-described preferred embodiments, these embodiments are merely illustrative and for illustrative purposes only. Based on these, various substitutions and modifications can be made to the application, all of which are included within the scope of protection.

Claims

1. A hypoxia-specific gene expression cassette, a) A promoter containing an FNR binding site, which is a forward hypoxic promoter whose expression can be induced under hypoxic conditions. b) Essential genes for survival, and c) A promoter comprising FNR and ArcA binding sites, comprising a reverse hyperoxygen promoter capable of functioning under conditions of normal organ oxygen content, The aforementioned survival-essential gene is a hypoxia-specific gene expression cassette, which encodes alanine racemase.

2. A hypoxia-specific gene expression cassette, a) A promoter containing an FNR binding site, which is a forward hypoxic promoter whose expression can be induced under hypoxic conditions. b) Essential genes for survival, and c) A promoter comprising two ArcA binding sites, comprising a reverse hyperoxygen promoter capable of functioning under conditions of normal organ oxygen content, The aforementioned survival-essential gene is a hypoxia-specific gene expression cassette, which encodes alanine racemase.

3. The FNR binding site of the forward hypoxia promoter or reverse hyperxia promoter matches the pattern TTGATNNNNNATCAA, where N is any base from A, T, C, and G. Any base in the TTGAT and ATCAA sequences at the conserved binding site is substituted, but the total number of substitutions is 3 or less, and three consecutive bases cannot be substituted. The hypoxia-specific gene expression cassette according to claim 1 or 2, wherein the ArcA binding site of the reverse hyperoxygen promoter matches the GTTAATTA pattern, and any base is substituted, but the total number of substitutions is 2 or less.

4. The forward hypoxia promoter is selected from the promoter region array of yhbU, ynfK, tdcA, yecH, or focA. Essential survival genes are selected from alfr or dadX. The hypoxia-specific gene expression cassette according to any one of the claims, wherein the reverse hyperoxygen promoter is selected from the promoter region sequences of cyoA, ydcI, phoH, argT, mqo, or lldP.

5. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene arlr, and a reverse hyperxia promoter cyoA.

6. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene alur, and a reverse hyperxia promoter ydcI.

7. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene alur, and a reverse hyperxia promoter phoH.

8. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene arlr, and a reverse hyperxia promoter argT.

9. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene alur, and a reverse hyperxia promoter mqo.

10. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene alur, and a reverse hyperxia promoter cyoA.

11. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene alur, and a reverse hyperxia promoter phoH.

12. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises the forward hypoxia promoter ynfK, the survival essential gene alur, and the reverse hyperxia promoter argT.

13. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene alur, and a reverse hyperxia promoter ydcI.

14. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene alur, and a reverse hyperxia promoter mqo.

15. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter tdcA, a survival essential gene alur, and a reverse hyperxia promoter ydcI.

16. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter tdcA, a survival essential gene alur, and a reverse hyperxia promoter cyoA.

17. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene arlr, and a reverse hyperxia promoter cyoA.

18. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene alur, and a reverse hyperxia promoter lldP.

19. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises the forward hypoxia promoter yecH, the survival essential gene arlr, and the reverse hyperxia promoter argT.

20. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene alur, and a reverse hyperxia promoter phoH.

21. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene alur, and a reverse hyperxia promoter mqo.

22. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter focA, a survival essential gene arlr, and a reverse hyperxia promoter lldP.

23. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter cyoA.

24. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter ydcI.

25. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter phoH.

26. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter argT.

27. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperxia promoter mqo.

28. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperxia promoter cyoA.

29. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperxia promoter phoH.

30. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperxia promoter argT.

31. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperxia promoter ydcI.

32. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperxia promoter mqo.

33. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter tdcA, a survival essential gene dadX, and a reverse hyperxia promoter ydcI.

34. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter tdcA, a survival essential gene dadX, and a reverse hyperxia promoter cyoA.

35. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene dadX, and a reverse hyperxia promoter cyoA.

36. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene dadX, and a reverse hyperxia promoter lldP.

37. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises the forward hypoxia promoter yecH, the survival essential gene dadX, and the reverse hyperxia promoter argT.

38. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene dadX, and a reverse hyperxia promoter phoH.

39. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter yecH, a survival essential gene dadX, and a reverse hyperxia promoter mqo.

40. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette comprises a forward hypoxia promoter focA, a survival essential gene dadX, and a reverse hyperxia promoter lldP.

41. The hypoxia-specific gene expression cassette according to any one of the claims, wherein the forward hypoxia promoter and / or reverse hyperxia promoter are promoters derived from Gram-negative bacteria.

42. Gram-negative bacteria include Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Chlonobacter, Klebsiella, Pantoea, Serratia, Simwellia, Enterobacter ludwigia, 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, Plague bacillus, and Neisseria cata. A hypoxia-specific gene expression cassette according to claim 41, selected from Helicobacter pylori, Moraxella catarrhalis, Campylobacter jejuni, Shigella, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Plague bacillus, Bacillus sonne, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori.

43. The hypoxia-specific gene expression cassette according to any one of the claims, wherein the forward hypoxia promoter is yhbU selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Chronobacter, Klebsiella, Pantoea, Serratia, and Simwellia.

44. The hypoxia-specific gene expression cassette according to any one of the claims, wherein the forward hypoxia promoter is ynfK selected from Salmonella, Escherichia coli, Serratia, Shigella, and Enterobacter dulwigia.

45. The hypoxia-specific gene expression cassette according to any one of the claims, wherein the alanine racemase gene alfr and / or dadX is selected from Salmonella, Escherichia coli, Shigella, Klebsiella, Yersinia, Haemophilus, and Pseudomonas.

46. A hypoxia-specific gene expression cassette according to any one of the claims, which is controlled by oxygen concentration.

47. The hypoxia-specific gene expression cassette according to claim 46, wherein the forward hypoxia promoter functions when the oxygen content is less than 1% and cannot function when the oxygen content is greater than 1%, and / or the reverse hyperxia promoter functions when the oxygen content is greater than 1% and cannot function when the oxygen content is less than 1%.

48. A hypoxia-specific gene expression cassette according to claim 47, wherein a forward hypoxia promoter functions when the oxygen content is less than 0.8% and cannot function when the oxygen content is greater than 0.8%, and / or a reverse hyperxia promoter functions when the oxygen content is greater than 0.8% and cannot function when the oxygen content is less than 0.8%.

49. A hypoxia-specific gene expression cassette according to any one of the claims, which is incorporated into the chromosome of a host bacterium.

50. A modified Gram-negative bacterium, which is modified by a hypoxia-specific gene expression cassette according to any one of the claims.

51. Gram-negative bacteria include Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Chronobacter, Klebsiella, Pantoea, Serratia, Simwellia, Enterobacter ludwigia, 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, Plague bacteria, and Nymphatica. The Gram-negative bacteria according to claim 50 are Ceria catarrhagicum, Moraxella catarrhalis, Campylobacter jejuni, Shigella, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Plague bacillus, Bacillus sonne, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori.

52. The Gram-negative bacterium described in claim 51, which is Salmonella.

53. A pharmaceutical composition comprising a hypoxia-specific gene expression cassette according to any one of claims 1 to 49, or a Gram-negative bacterium according to any one of claims 50 to 52.

54. The pharmaceutical composition according to claim 53, further comprising a pharmaceutically acceptable carrier.

55. The pharmaceutical composition according to claim 54, wherein the pharmaceutically acceptable carrier is selected from disintegrants, binders, fillers, buffers, tensioners, stabilizers, antioxidants, surfactants, and lubricants.

56. A pharmaceutical composition according to any one of claims 53 to 55, used for the treatment of solid tumors.

57. The use of a hypoxia-specific gene expression cassette according to any one of claims 1 to 49, a Gram-negative bacterium according to any one of claims 50 to 52, or a pharmaceutical composition according to any one of claims 53 to 56 in the preparation of an antitumor drug.

58. The use according to claim 57, wherein the tumor is a solid tumor.

59. The solid tumor is selected from tumors / cancers such as breast cancer, bone cancer, liver cancer, lung cancer, skin cancer, kidney cancer, stomach cancer, pancreatic cancer, prostate cancer, lymph node cancer (non-Hodgkin lymphoma, Hodgkin lymphoma), intestinal cancer (colon cancer, rectal cancer), pelvic cavity cancer (cervical cancer, ovarian malignant tumor, endometrial cancer, ovarian cancer), nervous system cancer, head and neck cancer, bladder cancer, etc., according to the pharmaceutical composition according to claim 56, or the use according to claim 58.

60. The pharmaceutical composition according to claim 56, or the use according to claim 56, 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.

61. A method for treating a tumor, comprising administering to a subject a hypoxia-specific gene expression cassette according to any one of claims 1 to 49, a Gram-negative bacterium according to any one of claims 50 to 52, or a pharmaceutical composition according to any one of claims 53 to 56.

62. The method according to claim 61, wherein the tumor is a solid tumor.

63. The method according to claim 62, wherein the solid tumor is selected from tumors / cancers such as breast cancer, bone cancer, liver cancer, lung cancer, skin cancer, kidney cancer, stomach cancer, pancreatic cancer, prostate cancer, lymph node cancer (non-Hodgkin lymphoma, Hodgkin lymphoma), intestinal cancer (colon cancer, rectal cancer), pelvic cavity cancer (cervical cancer, ovarian malignant tumor, endometrial cancer, ovarian cancer), nervous system cancer, head and neck cancer, and bladder cancer.

64. The method according to claim 63, 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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