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

Abstract

Provided are a hypoxia-specific gene expression cassette, Gram-negative bacteria containing the expression cassette, and a use of the Gram-negative bacteria in preparation of a drug for treating tumors and in treatment of tumors. By reasonably designing a forward hypoxia promoter and a reverse hyperoxia promoter, and coordinating with an appropriate survival-essential gene, a more efficient and safer hypoxia-specific gene expression cassette is obtained. The Gram-negative bacteria modified by the hypoxia-specific gene expression cassette obtained by design have a low overall mutation rate, and in a mouse in-vivo tumor model, more bacteria are distributed in a tumor and have a higher removal speed in normal organs.

Description

A hypoxia-specific gene expression cassette, bacteria containing the same, and applications thereof

[0001] This application claims priority to a prior application, patent application number 2023112106010, filed with the State Intellectual Property Office of China on September 19, 2023, entitled “A Hypoxia-Specific Gene Expression Cassette, Bacteria Containing the Same, and Applications thereof.” The entire text of that prior application is incorporated herein by reference. Technical Field

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

[0003] Among all malignant tumors, solid tumors account for about 90%, such as sarcoma, melanoma, breast cancer, lung cancer, colon cancer, prostate cancer, etc.

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

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

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

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

[0008] Studies have shown that after Salmonella typhimurium is attenuated using various methods, its ability to colonize tumor tissue can reach 1,000-10,000 times that of normal tissue. Methods for attenuating the strain include:

[0009] 1. AroA gene defect, strain SL7207. The aroA mutant strain is unable to synthesize para-aminobenzoic acid (PABA) and aromatic amino acids on its own, and the PABA content in the body environment is low, thereby reducing its toxicity in the body.

[0010] 2. Defects in the purI and msbB genes, strain VNP20009. The purI mutation prevents adenosine monophosphate synthesis, reducing virulence in vivo; the msbB mutation blocks lipid A synthesis in cell wall lipopolysaccharide, thereby preventing bacterial sepsis caused by excessive TNF-α secretion. In preliminary animal studies, this strain has been shown to inhibit tumor growth, and Phase I clinical trials have demonstrated its safety when administered intravenously as a live bacterium.

[0011] 3. Leucine and arginine deficiency, strains A1 and A1-R. Zhao et al. induced mutations and screened A1 using chemical reagents, and found that it was able to inhibit tumor cell growth in both in vivo and in vitro experiments. To enhance its tumor-targeting ability, Zhao et al. re-isolated A1 from within the tumor to obtain the A1-R strain with a stronger affinity for tumor tissue. In addition, Salmonella not only has the ability to inhibit tumor growth, but can also conveniently carry different DNA vectors. For example, Escherichia coli cytosine deaminase (CD), mitomycin C, and colicin E3 were all loaded into VNP20009 for therapeutic trials in various tumor models.

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

[0013] Salmonella YB1 is a tumor-targeting strain developed based on the above research of predecessors, combined with chromosome homologous recombination technology and the concept of synthetic biology. It has a preference for tumor hypoxic areas and tumor accumulation ability.

[0014] The specific method is to use the anaerobic promoter pepT in conjunction with the reverse oxygen promoter sodA to control the expression of the lethal gene asd, thereby enabling tumor survival and growth under hypoxic conditions and reducing accumulation in normal organs. This, in turn, increases safety compared to ordinary oncolytic bacteria.

[0015] YB1 is the first generation of attenuated Salmonella based on oxygen-regulated promoters designed from the analysis of oxygen in tumors. Therefore, from the perspective of its mechanism, it is significantly superior to ordinary nutritionally deficient strains. However, due to the characteristics of bacteria that are prone to mutation, it is necessary to pay attention to the mutation of bacteria under various pressures. In 2011, Dr. Yu Bin pointed out that YB1 is very easy to evolve in the absence of diaminopimelate (DAP), thereby breaking away from the regulation of oxygen. After research, it was found that the mutation frequency of YB1 to oxygen is as high as 10 -5 -10 -6Each bacterium. Studies have shown that the mutation frequency of Escherichia coli, which is the same enterobacterial genus as Salmonella, is 10 -9 -10 -10 Each generation of bacteria. Studies have also shown that the natural mutation frequency of Salmonella is 10 -8 Salmonella divides once every 20-30 minutes, which translates to a mutation frequency of YB1 that is 10,000 times higher than the natural mutation frequency of Salmonella. Therefore, the significant increase in YB1's mutation frequency is abnormal. This abnormally high mutation frequency reduces the advantages of hypoxia regulation, potentially affecting its safety and introducing new risks for cancer treatment. This may be due to irrational regulation of the genes involved: the pepT promoter, the asd gene, and the sodA promoter. Therefore, a new regulatory model is crucial.

[0016] According to the 2012 YB1 study, although the distribution of YB1 in normal organs of mice is on a downward trend, it is difficult to completely eliminate it in a short period of time. 3 -10 4 Distribution per gram of tissue. While it takes up to 26 days to completely clear most normal organs in mice, a small amount remains in the liver. This may be due to a high evolution rate caused by a design flaw in YB1 itself.

[0017] In summary, this field urgently needs a new generation of live bacterial strains that have efficient tumor targeting, rapid clearance capabilities in normal organs, and can also effectively inhibit tumor growth.

[0018] Summary of the Invention

[0019] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terminology and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the relevant fields and are standard procedures.

[0020] In view of the above-mentioned defects in the prior art, the present invention provides a hypoxia-specific gene expression cassette comprising:

[0021] a) a positive hypoxia promoter, which is a promoter comprising an FNR binding site, and the positive hypoxia promoter can be induced to express under hypoxia;

[0022] b) genes essential for survival; and

[0023] c) an inverted hyperoxic promoter, which is a promoter comprising FNR and ArcA binding sites, and the inverted hyperoxic promoter can function under the oxygen content conditions of normal organs;

[0024] The essential gene for survival is a gene encoding alanine racemase.

[0025] Another aspect of the present invention provides a hypoxia-specific gene expression cassette comprising:

[0026] a) a positive hypoxia promoter, which is a promoter comprising an FNR binding site, and the positive hypoxia promoter can be induced to express under hypoxia;

[0027] b) genes essential for survival; and

[0028] c) an inverted hyperoxic promoter, which is a promoter comprising two ArcA binding sites and capable of functioning under normal organ oxygen content conditions;

[0029] The essential gene for survival is a gene encoding alanine racemase.

[0030] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0031] In the present invention, "polynucleotide" refers to a macromolecule composed of multiple nucleotides linked by phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present invention can be codon-optimized for different host cells (e.g., Escherichia coli) to improve polypeptide expression. Methods for codon optimization are known in the art.

[0032] "Sequence identity" between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for assessing the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various known sequence analysis software. For example, sequence identity can be assessed using the EMBL-EBI online alignment tool (https: / / www.ebi.ac.uk / Tools / psa / ). The sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm using default parameters.

[0033] In order for bacteria to survive only in low oxygen concentrations, three requirements need to be met:

[0034] 1) Control the strength of the upstream promoter and background leakage expression.

[0035] If the oxygen sensor is partially leaky without binding to the upstream promoter, it will be impossible to regulate oxygen, resulting in the bacteria surviving at any oxygen concentration. Alternatively, if the upstream promoter is too weak, it will be unable to initiate the expression of downstream genes, resulting in the bacteria not surviving at any oxygen concentration.

[0036] 2) Select appropriate genes essential for survival.

[0037] By selecting genes essential for survival, it is possible to ensure that the bacteria die when the genes are not expressed, and to quickly ensure bacterial survival when the genes are induced by hypoxia;

[0038] 3) Under the switching conditions of low oxygen and high oxygen, the upstream promoter can quickly initiate transcription and quickly realize the synthetic expression of genes essential for survival, ensuring that the modified bacteria can survive. However, under high oxygen conditions, the expression of essential survival genes will not be initiated, resulting in bacterial death.

[0039] Therefore, in the present invention, a "hypoxia-specific gene expression cassette" refers to a segment of DNA that can initiate expression of an essential gene under hypoxic conditions, which contains an essential gene controlled by a hypoxia-inducible promoter and, if necessary, may further contain other regulatory elements required for the expression of the essential gene.

[0040] In the present invention, an "essential gene" refers to a gene that plays a crucial role in bacterial growth and / or survival. If the bacterium lacks this gene or its functional expression product, it will not survive, divide, and / or grow normally. Typical examples of bacteria lacking essential genes or their functional expression products are auxotrophic strains, which, in the absence of specific exogenous supplements, cannot survive, divide, and / or grow normally under in vitro culture conditions or in vivo environments. Essential genes are typically present in a single copy on the bacterial chromosome.

[0041] From this, we can see that the requirements for genes essential for survival are as follows:

[0042] (1) It is an essential gene for bacterial reproduction. If it is missing, it will lead to rapid death of bacteria;

[0043] (2) The product of this gene does not exist in the normal environment or the human body, which ensures that it will not get out of control in the human environment, and can be easily cultured and prepared by adding the corresponding expression product of this gene in a normal culture environment;

[0044] (3) This gene needs to be rapidly activated under the regulation of the hypoxia promoter and can quickly synthesize products to realize the regulatory function of the host bacteria.

[0045] In the present invention, the essential gene for survival is a gene encoding alanine racemase.

[0046] The cell wall is an essential component of Gram-negative bacteria such as Escherichia coli and Salmonella. The core component of the cell wall is peptidoglycan. To synthesize peptidoglycan, bacteria require D-alanine, a crucial building block. Without D-alanine, bacteria cannot synthesize their cell wall and subsequently undergo cell lysis.

[0047] All amino acids exist in nature are L-type, so there are two genes in Gram-negative bacteria: the alr gene for the biosynthesis of alanine racemase and dadX, which are responsible for converting L-alanine into D-alanine to meet the needs of cell wall synthesis.

[0048] The study found that if the alr gene and dadX gene mutate at the same time, a lethal mutation of Salmonella can be achieved, and this mutation can be compensated by additional supplementation of D-alanine in the culture medium.

[0049] In our previous research, YB1 Salmonella used the asd gene as an essential gene for regulation. Compared to YB1 Salmonella, the present invention selected the alr gene and dadX gene as essential genes for survival. The alr gene and dadX gene are functionally homologous genes. Therefore, the alanine racemase gene in the present invention can be the alr gene (SEQ ID No. 17) and the protein sequence of alr from Salmonella (SEQ ID No. 18), or the dadX gene (SEQ ID No. 19) and the protein sequence of dadX from Salmonella (SEQ ID No. 20); or the alr gene (SEQ ID No. 21) and the protein sequence of alr from E. coli (SEQ ID No. 22), or the dadX gene (SEQ ID No. 23) and the protein sequence of dadX from E. coli (SEQ ID No. 24); or the alr gene (SEQ ID No. 25) and the protein sequence of alr from Shigella (SEQ ID No. 26), or the dadX gene (SEQ ID No. 27) and the protein sequence of dadX from Shigella (SEQ ID No. 28); or the alr gene (SEQ ID No. 28) from Klebsiella. No. 29) and the protein sequence of Klebsiella alr (SEQ ID No. 30) or the Klebsiella dadX gene (SEQ ID No. 31) and the protein sequence of Klebsiella dadX (SEQ ID No. 32); or the Yersinia alr gene (SEQ ID No. 33) and the protein sequence of Yersinia alr (SEQ ID No. 34) or the Yersinia dadX gene (SEQ ID No. 35) and the protein sequence of Yersinia dadX (SEQ ID No. 36); or the Haemophilus alr gene (SEQ ID No. 37) and the protein sequence of Haemophilus alr (SEQ ID No. 38); or the Pseudomonas alr gene (SEQ ID No. 39) and the protein sequence of Pseudomonas alr (SEQ ID No. 40) or the Pseudomonas dadX gene (SEQ ID No. 41) and the protein sequence of Pseudomonas dadX (SEQ ID No. 42).

[0050] In the present invention, the alanine racemase alr gene and dadX gene comprise nucleotide sequences having a sequence identity of greater than or equal to 81%, preferably greater than or equal to 82%, more preferably greater than or equal to 83%, greater than or equal to 84%, greater than or equal to 85%, greater than or equal to 86%, greater than or equal to 87%, greater than or equal to 88%, greater than or equal to 89%, greater than or equal to 90%, greater than or equal to 91%, greater than or equal to 92%, greater than or equal to 93%, greater than or equal to 94%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, or most preferably greater than or equal to 99% to the above-mentioned specific sequences.

[0051] In this study, we first constructed bacteria with the alr gene knocked out. We then modified another gene, dadX, to create bacteria deficient in both alr and dadX. We then used the YB1 forward hypoxia promoter, pepT, and reverse hyperxia promoter, sodA, to regulate the additional alr gene (SWT006). Experimental results (SWT006 validation experiments) demonstrated that the alr and dadX genes, selected as essential genes, offer greater controllability than the asd gene, previously selected for YB1 Salmonella.

[0052] The positive hypoxic promoter described herein is a hypoxic promoter regulated by FNR; the reverse hyperoxic promoter described herein is an antisense promoter negatively regulated by FNR and / or ArcA. The fumarate and nitrate reduction genes, fnr, are important genes regulating aerobic and anaerobic growth in Salmonella. This complex regulatory system has been extensively studied in Escherichia coli and Salmonella. The DNA-binding protein FNR encoded by the fnr gene senses changes in oxygen and controls the expression of various genes, achieving overall metabolic shifts. Therefore, DNA-binding sequences such as FNR and ArcA have become important mechanisms for controlling downstream gene expression.

[0053] FNR has an oxygen-sensitive [4Fe-4S] 2+ The FNR domain directly senses oxygen and regulates site-specific DNA binding. In contrast, ArcA senses signals from the aerobic respiratory chain. Therefore, Gram-negative facultative anaerobes such as Salmonella and Escherichia coli utilize two distinct mechanisms for sensing changes in oxygen concentration. FNR regulates gene expression in response to hypoxia, either through activation or repression.

[0054] To this end, the present invention screened for the positive hypoxic promoters yhbU, ynfK, yecH, cydA, focA, and tdcA, which activate downstream gene expression under hypoxic conditions by FNR, and the negative hyperoxic promoters ydcI, cyoA, phoH, argT, mqo, and lldP, which inhibit downstream gene expression under hypoxic conditions by FNR and / or ArcA. Furthermore, promoters from different sources were screened as the positive hypoxic promoter and the negative hyperoxic promoter used in the present invention.

[0055] Among them, the positive hypoxia promoter used in the present invention is as follows:

[0056] (1) Salmonella yhbU promoter (yhbU-S) (SEQ ID No. 1), which contains the FNR binding site "CTGCCTTAAATCAA";

[0057] (2) Escherichia coli ynfK promoter (ynfK-E) (SEQ ID No. 2), wherein the FNR binding site is "TTGCGCTATCTCAA";

[0058] (3) Salmonella tdcA promoter (tdcA-S) (SEQ ID No. 3), which contains the FNR binding site "TTGATTGAAATCAG";

[0059] (4) Escherichia coli tdcA promoter (tdcA-E) (SEQ ID No. 4), which contains the FNR binding site "TTGACAAAAATCAG";

[0060] (5) Escherichia coli yecH promoter (yecH-E) (SEQ ID No. 5), which contains the FNR binding site "TTCATAAGCGGCAA";

[0061] (6) Salmonella ynfK promoter (ynfK-S) (SEQ ID No. 6), which contains the FNR binding site "TTGCGCTAACTCAA";

[0062] (7) Escherichia coli cydA promoter (cydA-E) (SEQ ID No. 7), wherein the FNR binding site is "TTGATATTTATCAA";

[0063] (8) Escherichia coli focA promoter (focA-E) (SEQ ID No. 8), wherein the FNR binding site is "ATGATCTATATCAA".

[0064] Among them, the reverse hyperoxia promoter used in the present invention is as follows:

[0065] (1) Salmonella cyoA promoter (cyoA-S) (SEQ ID No. 9), which contains the FNR binding site "TTTATTGATAATAA" and the ArcA binding site "GTTAAGTA";

[0066] (2) Salmonella phoH promoter (phoH-S) (SEQ ID No. 10), which contains the FNR binding site "TTGAGCGCCGTTAA" and the ArcA binding site "GTTAATTA";

[0067] (3) Salmonella lldP ​​promoter (lldP-S) (SEQ ID No. 11), which contains the FNR binding site "TCCATACACAACAA" and the ArcA binding site "GTTAACTA";

[0068] (4) Salmonella argT promoter (argT-S) (SEQ ID No. 12), which contains two ArcA binding sites, the first ArcA binding site is "TTTAATTC" and the second ArcA binding site is "TTTTATTA";

[0069] (5) Salmonella ydcI promoter (ydcI-S) (SEQ ID No. 13), which contains the FNR binding site "GTTATCAAAAACAA" and the ArcA binding site "GTTAATAA";

[0070] (6) Escherichia coli phoH promoter (phoH-E) (SEQ ID No. 14), which contains the FNR binding site "TTCATCACTGTCAT" and the ArcA binding site "GTTAAATA";

[0071] (7) Escherichia coli lldP ​​promoter (lldP-E) (SEQ ID No. 15), which contains the FNR binding site "TTCATTGTCATTAT" and the ArcA binding site "TTTAGTTA";

[0072] (8) Escherichia coli ydcI promoter (ydcI-E) (SEQ ID No. 16), which contains the FNR binding site "GTTATCATAATCAA" and the ArcA binding site "GTTAATAA";

[0073] (9) Escherichia coli mqo promoter (mqo) (SEQ ID No. 62), which contains two ArcA binding sites, the first ArcA binding site is "ATTATTTA" and the second ArcA binding site is "GTTACTTA".

[0074] Through analysis of the above-mentioned reverse hypoxia promoter and forward hyperoxia promoter, we found that the FNR binding sites all conform to the "TTGATNNNNATCAA" pattern, and any base in the TTGAT and ATCAA sequences in the conserved binding sites can be replaced, but the total number does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; and ArcA conforms to the "GTTAATTA" pattern of its core region, and any base in the GTTAATTA sequence in the conserved binding site can be replaced, but the total number does not exceed 2.

[0075] Therefore, in a preferred embodiment of the present invention, the FNR binding site of the forward hypoxia promoter or the reverse hyperoxia promoter conforms to the pattern of TTGATNNNNATCAA, where N is any base of A, T, C, or G, and any base in the TTGAT or ATCAA sequence in the conserved binding site can be replaced, but the total number of replacements does not exceed 3, and three consecutive adjacent bases cannot be replaced; the ArcA binding site of the reverse hyperoxia promoter conforms to the pattern of GTTAATTA, wherein any base can be replaced, but the total number of replacements does not exceed 2.

[0076] 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, dadX; and the reverse hyperoxia promoter is selected from cyoA, ydcI, phoH, argT, mqo, lldP.

[0077] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter cyoA.

[0078] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter ydcI.

[0079] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter phoH.

[0080] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter argT.

[0081] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter mqo.

[0082] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene alr and the reverse hyperoxia promoter cyoA.

[0083] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene alr and the reverse hyperoxia promoter phoH.

[0084] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene alr and the reverse hyperoxia promoter argT.

[0085] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene alr and the reverse hyperoxia promoter ydcI.

[0086] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene alr and the reverse hyperoxia promoter mqo.

[0087] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter tdcA, the survival essential gene alr and the reverse hyperoxia promoter ydcI.

[0088] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter tdcA, the survival essential gene alr and the reverse hyperoxia promoter cyoA.

[0089] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yecH, the survival essential gene alr and the reverse hyperoxia promoter cyoA.

[0090] In a further preferred embodiment of the present invention, the expression cassette consists of a forward hypoxia promoter yecH, a survival essential gene alr and a reverse hyperoxia promoter lldP.

[0091] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yecH, the survival essential gene alr and the reverse hyperoxia promoter argT.

[0092] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yecH, the survival essential gene alr and the reverse hyperoxia promoter phoH.

[0093] In a further preferred embodiment of the present invention, the expression cassette consists of a forward hypoxia promoter yecH, a survival essential gene alr and a reverse hyperoxia promoter mqo.

[0094] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter focA, the survival essential gene alr and the reverse hyperoxia promoter lldP.

[0095] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene dadX and the reverse hyperoxia promoter cyoA.

[0096] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene dadX and the reverse hyperoxia promoter ydcI.

[0097] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene dadX and the reverse hyperoxia promoter phoH.

[0098] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene dadX and the reverse hyperoxia promoter argT.

[0099] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene dadX and the reverse hyperoxia promoter mqo.

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

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

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

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

[0104] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene dadX and the reverse hyperoxia promoter mqo.

[0105] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter tdcA, the survival essential gene dadX and the reverse hyperoxia promoter ydcI.

[0106] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter tdcA, the survival essential gene dadX and the reverse hyperoxia promoter cyoA.

[0107] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yecH, the survival essential gene dadX and the reverse hyperoxia promoter cyoA.

[0108] In a further preferred embodiment of the present invention, the expression cassette consists of a forward hypoxia promoter yecH, a survival essential gene dadX and a reverse hyperoxia promoter lldP.

[0109] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yecH, the survival essential gene dadX and the reverse hyperoxia promoter argT.

[0110] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yecH, the survival essential gene dadX and the reverse hyperoxia promoter phoH.

[0111] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yecH, the survival essential gene dadX and the reverse hyperoxia promoter mqo.

[0112] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter focA, the survival essential gene dadX and the reverse hyperoxia promoter lldP.

[0113] The positive hypoxia promoter disclosed in patent CN104471057B is pepT. The pepT promoter is not completely regulated by FNR, but is a promoter that is dual-regulated by CRP-cAMP and FNR. Therefore, half of the FNR binding site in the pepT promoter is a CRP binding site, and half is a FNR binding site. The CRP-FNR binding region sequence of the pepT promoter is GTGACCTGACGCAA, of which the first half GTGA conforms to the first half of the CRP conserved binding site GTGANNNNNNTCAC, and the second half CGCAA conforms to the ATCAA portion of the FNR conserved region. The A at the tenth position is replaced by C, and the T at the eleventh position is replaced by G, which does not conform to the rules for positive hypoxia promoters in the present invention.

[0114] The FNR binding region of the reverse hyperoxia promoter sodA, disclosed in patent CN104471057B, contains only the first half of the FNR region, TTGAT, while the second half contains three consecutive substitutions of ATCAA with ATTTT, thus not containing a complete FNR binding site. The ArcA binding site is TTTAATTA, which, compared to the conserved core ArcA binding site 5'-GTTAATTA-3', has the first G replaced by a T. Therefore, the sodA promoter contains only a single ArcA binding site, and its FNR binding site is incomplete, which does not meet the requirements of the present invention for reverse hyperoxia promoters.

[0115] In summary, the present invention obtains a more efficient and safer hypoxia-specific gene expression cassette by screening a reasonable forward hypoxia promoter and a reverse hyperoxia promoter, and combining them with appropriate survival essential genes.

[0116] In a preferred embodiment of the present invention, the forward hypoxic promoter and / or the reverse hyperoxic promoter is a promoter derived from Gram-negative bacteria.

[0117] In the present invention, term " gram-negative bacteria " refers to after carrying out the known partial program of gram staining, does not retain the bacterium of the initial basic dye staining agent (such as, crystal violet) as.In exemplary gram staining, first by heating, cell is fixed on slide and dyed with basic dye (such as, crystal violet), and described basic dye is absorbed by both gram-negative bacteria and gram-positive bacteria.Then, slide is processed with mordant (such as, Gram's iodine solution), and described mordant is incorporated into basic dye (such as, crystal violet) and is trapped in cell.Then, by cell acetone or ethanol washing, then with the second dye of different colors counterstaining (such as, safranin).Gram-positive organism retains initial purple dyeing, and gram-negative organism decolorizes by organic washing solvent, therefore shows counterstaining. Exemplary Gram-negative bacteria include, but are not limited to, Escherichia species, Shigella species, Salmonella species, Campylobacter species, Neisseria species, Haemophilus species, Aeromonas species, Francisella species, Yersinia species, Klebsiella species, Bordetella species, Legionella species, Corynebacterium species, Citrobacter species, Chlamydia species, Brucella species, Pseudomonas species, Helicobacter species, and Vibrio species.

[0118] Thus, in a more preferred embodiment of the present invention, the Gram-negative bacteria are selected from the group consisting of Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simihuierlia, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, Paralytica, Vibrio sanguineus, Vibrio cholerae, Yersinia pestis, Coccus catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella catarrhalis, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolytica, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia and Helicobacter pylori.

[0119] In the present invention, the term "live bacteria" refers to a strain that has vitality, active nutritional metabolic characteristics, and is able to perform its own biological functions. Live bacteria can include bacterial biomass produced during the metabolic process of the strain.

[0120] In a further preferred embodiment of the present invention, the positive hypoxia promoter is yhbU, which is selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia and Simihuiella.

[0121] In a further preferred embodiment of the present invention, the positive hypoxic promoter is ynfK, which is selected from the group consisting of Salmonella, Escherichia coli, Serratia, Shigella and Enterobacter reuteri.

[0122] In a preferred embodiment of the present invention, the alanine racemase gene alr and / or dadX is selected from Salmonella, Escherichia coli, Shigella, Klebsiella, Yersinia, Haemophilus and Pseudomonas.

[0123] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is regulated by oxygen concentration.

[0124] For Gram-negative bacteria like Salmonella to survive in a hypoxic zone, an oxygen concentration of 1% is crucial, representing pathological hypoxia. This is because oxygen concentrations below 1% are a clear marker of a tumor hypoxic zone. In normal organs and tissues, there are no areas with oxygen concentrations below 1%. For example, in pancreatic cancer, cervical cancer, prostate cancer, and other tumors, the oxygen concentration in the hypoxic zone is less than 0.7%.

[0125] The hypoxia-specific gene expression cassette designed in this invention precisely regulates oxygen levels, enabling Salmonella and other Gram-negative bacteria to recognize and proliferate in hypoxic tumor areas. The goal of this invention is to enable the modified Salmonella and other Gram-negative bacteria to survive and proliferate in oxygen concentrations below 0.8%, and to achieve suicide lysis in normal oxygen concentrations.

[0126] Thus, in a more preferred embodiment of the present invention, the positive hypoxia promoter functions when the oxygen content is below 1%, but fails to function when the oxygen content is above 1%; and / or

[0127] The inverted hyperoxic promoter is functional when the oxygen content is above 1%, but is inactive when the oxygen content is below 1%.

[0128] In a further preferred embodiment of the present invention, the positive hypoxia promoter functions when the oxygen content is lower than 0.8%, but fails to function when the oxygen content is higher than 0.8%; and / or

[0129] The inverted hyperoxic promoter is functional when the oxygen content is above 0.8%, but is inactive when the oxygen content is below 0.8%.

[0130] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is integrated into the chromosome of the host bacteria.

[0131] Another aspect of the present invention provides a modified Gram-negative bacterium, which is modified by the hypoxia-specific gene expression cassette of the present invention.

[0132] In a preferred embodiment of the present invention, the Gram-negative bacteria are Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simihuierlia, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, Parahaemolyticus Vibrio, Vibrio cholerae, Yersinia pestis, Coccus catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella catarrhalis, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolytica, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, or Helicobacter pylori.

[0133] In a more preferred embodiment of the present invention, the Gram-negative bacteria is Salmonella typhimurium.

[0134] Another aspect of the present invention provides use of the hypoxia-specific gene expression cassette of the present invention or the Gram-negative bacteria of the present invention in the preparation of tumor drugs.

[0135] In a preferred embodiment of the present invention, the tumor is a solid tumor.

[0136] The term "solid tumor" used in the present invention refers to an abnormal mass of tissue, generally not comprising a cyst or liquid area. Solid tumors may be benign (non-cancer), or malignant (cancer). Different types of malignant solid tumors are named after the cell type that forms them. Examples of malignant solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood cancer) does not form malignant solid tumors generally. Malignant solid tumors include but are not limited to the abnormal cell mass that may be derived from different tissue types, such as liver, colon, colorectal, skin, breast, pancreas, cervix uteri, uterine body, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testis, thyroid or lung etc., so malignant solid tumors include malignant solid liver tumors, colon tumors, colorectal tumors, skin tumors, breast tumors, pancreatic tumors, cervix tumors, uterine body tumors, bladder tumors, gallbladder tumors, kidney tumors, laryngeal tumors, lip tumors, oral cavity tumors, esophageal tumors, ovarian tumors, prostate tumors, stomach tumors, testicular tumors, thyroid tumors or lung tumors etc.

[0137] Thus, in a more preferred embodiment of the present invention, the solid tumor is selected from tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestine (colon cancer, rectal cancer), pelvic cavity (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer and bladder, etc.

[0138] In a further preferred embodiment of the present invention, the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

[0139] In the present invention, for the purpose of treatment, the term "subject" is preferably a subject in need of treatment for a target pathological condition, such as a tumor. For the purpose of prevention, the subject is preferably a subject at risk of developing a target pathological condition or prone to developing a target pathological condition. The term "subject" includes living organisms, such as prokaryotes and eukaryotes. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, hedgehogs, rats, and transgenic non-human animals. In a specific embodiment of the present invention, the subject is a human.

[0140] As used herein, "treatment" is a process for obtaining a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing the proliferation of neoplastic or cancerous cells (or destroying neoplastic or cancerous cells), inhibiting the metastasis of neoplastic cells, shrinking or reducing the size of a tumor, alleviating a malignant tumor, alleviating symptoms caused by a malignant tumor, improving the quality of life of a subject suffering from a malignant tumor, reducing the dosage of other drugs required to treat a malignant tumor, delaying the progression of a malignant tumor, curing a malignant tumor, and / or prolonging the survival of a patient suffering from a malignant tumor.

[0141] As used herein, an "effective amount" or "effective dose" of a bacterium, drug, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes that present during the course of disease. For therapeutic uses, beneficial or desired results include, for example, alleviating one or more symptoms of a disease (such as a tumor), reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, prolonging the survival of the treated subject, and / or delaying the progression of cancer in a patient. For example, an "effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in animal model systems that are predictive of efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Such an amount can be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0142] In some embodiments of the invention, the effective amount of bacteria in the bacteria, medicament or pharmaceutical composition comprises at least about 10 4 colony forming units (cfu), for example at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13 cfu, including any ranges between listed values, such as 10 4 -10 8 cfu, 10 4 -10 9 cfu, 10 4 -10 10 cfu, 10 4 -10 11 cfu, 10 4 -10 12 cfu, 10 4 -10 13cfu, 10 5 -10 8 cfu, 10 5 -10 9 cfu, 10 5 -10 10 cfu, 10 5 -10 11 cfu, 10 5 -10 12 cfu, 10 5 -10 13 cfu, 10 6 -10 8 cfu, 10 6 -10 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 cfu.

[0143] In some embodiments of the invention, the volume of an effective dose of the bacterium, medicament or pharmaceutical composition of the invention is less than or equal to about 3 ml, about 2.5 ml, about 2 ml, about 1.5 ml, about 1 ml, about 0.75 ml, about 0.5 ml, about 0.25 ml or about 0.1 ml. In some embodiments, the volume of an effective dose of the bacterium, medicament or pharmaceutical composition is about 20 ml, about 19 ml, about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml, about 13 ml, about 12 ml, about 11 ml, about 10 ml, about 9 ml, about 8 ml, about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml or about 1 ml. Optionally, the volume of the effective dose of the bacteria, drug or pharmaceutical composition is about 20.5 ml, about 19.5 ml, about 18.5 ml, about 17.5 ml, about 16.5 ml, about 15.5 ml, about 14.5 ml, about 13.5 ml, about 12.5 ml, about 11.5 ml, about 10.5 ml, about 9.5 ml, about 8.5 ml, about 7.5 ml, about 6.5 ml, about 5.5 ml, about 4.5 ml, about 3.5 ml, about 2.5 ml, about 1.5 ml, or about 0.5 ml. In some embodiments, the volume of an effective dose of the bacteria, drug or pharmaceutical composition is about 200 ml, about 190 ml, about 180 ml, about 170 ml, about 160 ml, about 150 ml, about 140 ml, about 130 ml, about 120 ml, about 110 ml, about 100 ml, about 90 ml, about 80 ml, about 70 ml, about 60 ml, about 50 ml, about 40 ml or about 30 ml, as well as ranges between any of the listed values, for example, 100 ml-110 ml, 100 ml-120 ml, 90 ml-120 ml, 90 ml-130 ml, etc. Optionally, the volume of the effective dose of the bacteria, drug or pharmaceutical composition is about 205 ml, about 195 ml, about 185 ml, about 175 ml, about 165 ml, about 155 ml, about 145 ml, about 135 ml, about 125 ml, about 115 ml, about 105 ml, about 95 ml, about 85 ml, about 75 ml, about 65 ml, about 55 ml, about 45 ml, about 35 ml or about 25 ml, and ranges between any of the listed values, such as 105 ml-115 ml, 105 ml-125 ml, 95 ml-125 ml, 95 ml-135 ml, etc.Optionally, the volume of an effective dose of the bacteria, drug or pharmaceutical composition is about 900 microliters, about 800 microliters, about 700 microliters, about 600 microliters, about 500 microliters, about 400 microliters, about 300 microliters, about 200 microliters, or about 100 microliters, optionally about 950 microliters, about 850 microliters, about 750 microliters, about 650 microliters, about 550 microliters, about 450 microliters, about 350 microliters, about 250 microliters, about 150 microliters, or about 50 microliters.

[0144] In some embodiments, the effective dose of the bacterium, drug, or pharmaceutical composition has a volume of less than or equal to about 2.0 ml.

[0145] The dosage ranges mentioned herein are merely exemplary and do not limit the dosage ranges that can be selected by a physician. The amount of the active ingredient (e.g., the bacteria of the present invention) in the pharmaceutical compositions of the present invention can vary depending on factors such as the individual's disease state, age, sex, and weight, including the presence or absence of a tumor, the type of tumor being treated, the severity of the tumor, the activity or viability of the bacteria, drug, or pharmaceutical composition, the route of administration, the duration of treatment, the drugs used in combination with the bacteria, drug, or pharmaceutical composition (if any), the subject's diet and general health, and similar factors well known in the art. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, a single dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased depending on the urgency of the therapeutic situation.

[0146] As used herein, a "pharmaceutically acceptable carrier" includes any material that allows the ingredient to retain biological activity when combined with the active ingredient and does not react with the subject's immune system, including but not limited to disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants, or lubricants. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion). For example, depending on the route of administration, the bacteria of the present invention can be wrapped in a material to protect the bacteria from the effects of acids and other natural conditions that can inactivate the bacteria. Pharmaceutically acceptable carriers include physiological saline, PBS buffer, sterile aqueous solutions or dispersions, and powders for the temporary preparation of injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any incompatibility with the active compound, may be in the pharmaceutical composition of the present invention.

[0147] Thus, 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 viable bacteria.

[0148] In a preferred embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0149] In a more preferred embodiment of the present invention, the pharmaceutically acceptable carrier is selected from disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants and lubricants.

[0150] In a preferred embodiment of the present invention, the pharmaceutical composition is used to treat solid tumors. The bacteria, drugs, or pharmaceutical compositions of the present invention are administered by intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0151] In a preferred embodiment of the present invention, the bacteria, drugs or pharmaceutical compositions of the present invention can be formulated for administration by the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0152] The dosage form of the drug or pharmaceutical composition of the present invention can be a solution, emulsion, freeze-dried preparation or suspension; for oral administration, the dosage form can be a tablet or capsule; for intranasal dosage form, the dosage form can be a powder, nasal drops or aerosol form; for topical administration, the dosage form can be an aqueous solution, suspension, ointment, cream or gel; for rectal or vaginal administration, the dosage form can be a suppository, enema or delivered as part of an endoscopic or colonoscopy procedure.

[0153] Bacteria, medicine or pharmaceutical compositions of the present invention can be manufactured by methods well known in the art, such as growing the microorganisms in a fermentation tank, followed by centrifugal concentration and washing, filtering or dialysis, conventional granulation, mixing, dissolving, encapsulating, lyophilizing or emulsifying processes and other methods. Bacteria, medicine or pharmaceutical compositions of the present invention can be produced in various forms, including particles, precipitations or microparticles, powders, including freeze-dried, rotary-dried or spray-dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. Preparations can optionally contain stabilizers, pH adjusting agents, surfactants, bioavailability regulators and combinations thereof.

[0154] The bacterium, medicine or pharmaceutical composition of the present invention can be administered alone or in combination with other compounds or compositions in the presence of a carrier. In a preferred embodiment of the present invention, the bacterium, medicine or pharmaceutical composition can be administered in combination with other malignant tumor therapies (including but not limited to, radiotherapy, chemotherapy and surgery). The bacterium, medicine or pharmaceutical composition can be used as an adjuvant in such a case.

[0155] In another aspect, the present invention provides a method for treating tumors, comprising administering the hypoxia-specific gene expression cassette of the present invention, the Gram-negative bacteria of the present invention, or the pharmaceutical composition of the present invention to a subject.

[0156] In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to treat tumors. In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to induce an anti-tumor specific immune response in a subject with a tumor. In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to induce anti-tumor immune memory in a subject with a tumor. In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to prevent or treat metastasis or recurrence of a tumor. In one embodiment, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is used to treat tumors that have become resistant to or failed previous anti-tumor therapies.

[0157] In one embodiment of the present invention, the tumor is a tumor of the nervous system, respiratory system, digestive system, urinary system, reproductive system, hematopoietic system, lymphatic system, endocrine system, or skin and mucosa. In one embodiment, the tumor is a sarcoma or carcinoma. In one embodiment, the tumor is a solid tumor.

[0158] In a further preferred embodiment of the present invention, the solid tumor is selected from the group consisting of breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal (colon cancer, rectal cancer), pelvic (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer and bladder tumors / cancers, etc.

[0159] In a more preferred embodiment of the present invention, the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

[0160] The method for treating tumors provided by the present invention 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.

[0161] In an embodiment of the present invention, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention can be administered by the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0162] In some embodiments of the invention, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the invention can be administered, for example, at intervals of about 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1 day, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including ranges between any two of the listed values, 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 24 hours. minutes-1 day, 10 minutes-30 minutes, 10 minutes-1 hour, 10 minutes-2 hours, 10 minutes-4 hours, 10 minutes-12 hours, 10 minutes-18 hours, 10 minutes-1 day, 30 minutes-1 hour, 30 minutes-2 hours, 30 minutes-4 hours, 30 minutes-12 hours, 30 minutes-18 hours, 30 minutes-1 day, 30 minutes-2 days, 1 hour-2 hours, 1 hour-4 hours, 1 hour-12 hours, 1 hour-18 hours, 1 hour-1 day, 4 hours-12 hours, 4 hours-18 hours, 4 hours-1 day, 1 day-2 days, 1 day-3 days, 1 day-4 days, 1 day-5 days, 1 day-7 days, 1 day-10 days, 2 days-3 days, 2 days-4 days, 2 days-5 days, 2 days-7 days, 2 days-10 days, or 5 days-10 days. In some embodiments, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks, or once every twenty-six weeks.In some embodiments, the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition is formulated, for example, to be administered at intervals of about 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1 day, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including ranges between any two of the listed values, 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 day to 24 hours. minutes-1 day, 10 minutes-30 minutes, 10 minutes-1 hour, 10 minutes-2 hours, 10 minutes-4 hours, 10 minutes-12 hours, 10 minutes-18 hours, 10 minutes-1 day, 30 minutes-1 hour, 30 minutes-2 hours, 30 minutes-4 hours, 30 minutes-12 hours, 30 minutes-18 hours, 30 minutes-1 day, 30 minutes-2 days, 1 hour-2 hours, 1 hour-4 hours, 1 hour-12 hours, 1 hour-18 hours, 1 hour-1 day, 4 hours-12 hours, 4 hours-18 hours, 4 hours-1 day, 1 day-2 days, 1 day-3 days, 1 day-4 days, 1 day-5 days, 1 day-7 days, 1 day-10 days, 2 days-3 days, 2 days-4 days, 2 days-5 days, 2 days-7 days, 2 days-10 days, or 5 days-10 days. In some embodiments, the bacteria, drug or pharmaceutical composition is formulated to be administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks or once every twenty-six weeks. The dosage regimen may depend on the pattern of pharmacokinetic decay that the practitioner expects to achieve. The progress of the therapy can be monitored by conventional techniques and assays. The dosage regimen can vary over time.

[0163] The present invention also provides a method for inducing an anti-tumor specific immune response in a subject having a tumor, comprising administering to the subject having a tumor an effective amount of the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention.

[0164] The present invention also provides a method for inducing anti-tumor immune memory in a subject having a tumor, comprising administering to the subject having a tumor an effective amount of the hypoxia-specific gene expression cassette, Gram-negative bacteria, or pharmaceutical composition of the present invention.

[0165] The present invention also 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 suffering from a tumor.

[0166] The present invention also 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 suffering from tumor metastasis or recurrence or a subject at high risk of tumor metastasis or recurrence.

[0167] The present invention also provides a method for treating a tumor that has become resistant to or failed to respond to previous anti-tumor therapy, 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 become resistant to or failed to respond to previous anti-tumor therapy.

[0168] The present invention achieves the following beneficial effects by adopting the above technical solution:

[0169] 1. The present invention obtains a more efficient and safer hypoxia-specific gene expression cassette by rationally designing a forward hypoxia promoter and a reverse hyperoxia promoter, and combining them with appropriate survival essential genes.

[0170] 2. The present invention selects alr or dadX as the essential genes for survival. The controllability of alr and dadX as essential genes for survival is higher than the essential gene asd selected by YB1 Salmonella.

[0171] 3. The hypoxia-specific gene expression cassette designed in this invention precisely regulates oxygen, enabling the modified bacteria to identify and proliferate in hypoxic tumor areas. The modified bacteria can survive and proliferate in oxygen concentrations below 0.8%, and can also undergo suicide lysis in normal oxygen concentrations.

[0172] 4. The bacteria modified with the hypoxia-specific gene expression cassette designed by the present invention have a low overall mutation rate. In a mouse tumor model, the modified bacteria are more distributed inside the tumor and are cleared faster from normal organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1. Validation of the positive hypoxia promoter clone library identified by agarose gel electrophoresis.

[0174] (A) Forward hypoxia promoters yhbU-S, tdcA-S, ynfK-S, ynfk-E, tdcA-E, yecH-E, cydA-E, and focA-E;

[0175] (B) Forward hypoxia promoter yhbU-E.

[0176] Figure 2. Amplification verification diagram of essential gene library identified by agarose gel electrophoresis.

[0177] Figure 3. Validation of the reverse hyperoxia promoter clone library identified by agarose gel electrophoresis.

[0178] (A) Reverse hyperoxia promoters cyoA-S, phoH-S, lldP-S, argT-S, ydcI-S, phoH-E, lldP-E, ydcI-E;

[0179] (B) Reverse expression of the hyperoxia promoter mqo.

[0180] Figure 4 Schematic diagram of the hypoxia-specific gene expression cassette.

[0181] Figure 5 Schematic diagram of the lambda RED recombinase and CRE recombinase systems.

[0182] Figure 6 shows the knockout experiment and identification of the Salmonella aroA gene.

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

[0184] (B) Verification of the PCR amplification product of the target fragment of strain SWT003 identified by agarose gel electrophoresis.

[0185] Figure 7. Knockout experiment and identification of Salmonella alr.

[0186] (A) Agarose gel electrophoresis to identify the PCR amplification product of the alr gene knockout fragment;

[0187] (B) Verification of the PCR amplification product of the target fragment of strain SWT004 identified by agarose gel electrophoresis.

[0188] FIG8 is a schematic diagram of the construction of plasmid pSWT004 used to construct YB1-like Salmonella SWT005.

[0189] FIG9 is a diagram showing the verification of enzyme digestion products of plasmid pSWT004 identified by agarose gel electrophoresis.

[0190] Figure 10 Verification of the PCR amplification product of cm-pepT-asd-sodA fragment identified by agarose gel electrophoresis.

[0191] FIG11 is a diagram showing verification of PCR amplification products of Salmonella SWT005 identified by agarose gel electrophoresis.

[0192] Figure 12 shows the results of the oxygen adaptability verification test of YB1-like Salmonella SWT005.

[0193] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0194] (B) Cultured under aerobic conditions on LB plates without D-alanine.

[0195] FIG13 is a diagram showing the verification of the PCR amplification product of plasmid pSWT005 identified by agarose gel electrophoresis.

[0196] FIG14 is a diagram showing the verification of the PCR amplification product of strain SWT006 identified by agarose gel electrophoresis.

[0197] Figure 15 shows the results of the oxygen adaptability verification test of the SWT006 strain.

[0198] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0199] (B) Cultured under aerobic conditions on LB plates without D-alanine.

[0200] FIG16 is a diagram of the strain verification library after insertion of the hypoxia-specific gene expression cassette (alr is an essential gene) as identified by agarose gel electrophoresis.

[0201] (A)SWT1001, SWT1002, SWT1003, SWT1004, SWT1005, SWT1006, SWT1007, SWT1008;

[0202] (B)SWT1009, SWT1010, SWT1011, SWT1012, SWT1013, SWT1014, SWT1015, SWT1016;

[0203] (C)SWT1017, SWT1018, SWT1019, SWT1020, SWT1021, SWT1022, SWT1023, SWT1024;

[0204] (D)SWT1025, SWT1026, SWT1027, SWT1028, SWT1029, SWT1030, SWT1031, SWT1032;

[0205] (E)SWT1033, SWT1034, SWT1035, SWT1036, SWT1037, SWT1038, SWT1039, SWT1040;

[0206] (F)SWT1041, SWT1042, SWT1043, SWT1044, SWT1045, SWT1046, SWT1047, SWT1048;

[0207] (G)SWT1049, SWT1050, SWT1051, SWT1052, SWT1053, SWT1054, SWT1055, SWT1056;

[0208] (H)SWT1057, SWT1058, SWT1059, SWT1060, SWT1061, SWT1062, SWT1063, SWT1064;

[0209] (I) SWT1065, SWT1066, SWT1067, SWT1068, SWT1069, SWT1070, SWT1071, SWT1072;

[0210] (J)SWT1073, SWT1074, SWT1075.

[0211] FIG17 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1001.

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

[0213] (B) Aerobic culture on LB plates without D-alanine.

[0214] FIG18 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1002.

[0215] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0216] (B) Aerobic culture on LB plates without D-alanine.

[0217] FIG19 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1004.

[0218] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0219] (B) Aerobic culture on LB plates without D-alanine.

[0220] FIG20 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1005.

[0221] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0222] (B) Aerobic culture on LB plates without D-alanine.

[0223] Figure 21 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1009.

[0224] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0225] (B) Aerobic culture on LB plates without D-alanine.

[0226] FIG22 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1010.

[0227] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0228] (B) Aerobic culture on LB plates without D-alanine.

[0229] FIG23 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1012.

[0230] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0231] (B) Aerobic culture on LB plates without D-alanine.

[0232] FIG24 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1013.

[0233] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0234] (B) Aerobic culture on LB plates without D-alanine.

[0235] Figure 25 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1021.

[0236] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0237] (B) Aerobic culture on LB plates without D-alanine.

[0238] FIG26 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1025.

[0239] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0240] (B) Aerobic culture on LB plates without D-alanine.

[0241] FIG27 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1033.

[0242] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0243] (B) Aerobic culture on LB plates without D-alanine.

[0244] FIG28 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1035.

[0245] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0246] (B) Aerobic culture on LB plates without D-alanine.

[0247] FIG29 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1036.

[0248] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0249] (B) Aerobic culture on LB plates without D-alanine.

[0250] Figure 30 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1038.

[0251] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0252] (B) Aerobic culture on LB plates without D-alanine.

[0253] FIG31 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1063.

[0254] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0255] (B) Aerobic culture on LB plates without D-alanine.

[0256] FIG32 shows the results of oxygen adaptability verification tests of various Salmonella strains at oxygen concentrations below 0.8%.

[0257] (A) SWT1001 was cultured on LB plates;

[0258] (B) SWT1005 was cultured on LB plates;

[0259] (C) SWT1009 was cultured on LB plates;

[0260] (D) SWT1013 was cultured on LB plates.

[0261] FIG33 Immunohistochemical identification of the distribution of Salmonella containing a hypoxia-specific gene expression cassette in the hypoxic area of ​​the tumor.

[0262] (A) Tumor section stained with anti-Salmonella antibody. The brown part indicated by the arrow is the antibody-positive area.

[0263] (B) Tumor section stained with anti-HIF1α antibody. The brown part indicated by the arrow is the antibody-positive area.

[0264] Figure 34 shows the invasion effect of strain SWT1001 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0265] (A) Effect on invasion of CT26 cell line;

[0266] (B) Effect on invasion of EMT6 cell line;

[0267] (C) Invasion effect on MB49 cell line;

[0268] (D) Invasion effect on RM-1 cell line;

[0269] (E) Invasion effect on A549 cell line;

[0270] (F) Invasion effect on K7M2 cell line;

[0271] (G) Invasion effect on B16F10 cell line;

[0272] (H) Invasion effect on MFC cell lines;

[0273] (I) Invasion effect on SCC7 cell line;

[0274] (J) Invasion effect on Hepa1-6 cell line;

[0275] (K) Invasion effect on Pan02 cell line;

[0276] (L) Invasion effect on ID8 cell line.

[0277] Figure 35 supplements Figure 34 , showing the invasion effect of strain SWT1001 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0278] (M) Invasion effect on Renca cell line;

[0279] (N) Invasion effect on Neuro-2a cell line.

[0280] Figure 36 shows the invasion effect of strain SWT1002 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0281] (A) Effect on invasion of CT26 cell line;

[0282] (B) Effect on invasion of EMT6 cell line;

[0283] (C) Invasion effect on MB49 cell line;

[0284] (D) Invasion effect on RM-1 cell line;

[0285] (E) Invasion effect on A549 cell line;

[0286] (F) Invasion effect on K7M2 cell line;

[0287] (G) Invasion effect on B16F10 cell line;

[0288] (H) Invasion effect on MFC cell lines;

[0289] (I) Invasion effect on SCC7 cell line;

[0290] (J) Invasion effect on Hepa1-6 cell line;

[0291] (K) Invasion effect on Pan02 cell line;

[0292] (L) Invasion effect on ID8 cell line.

[0293] Figure 37 supplements Figure 36 , showing the invasion effect of strain SWT1002 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0294] (M) Invasion effect on Renca cell line;

[0295] (N) Invasion effect on Neuro-2a cell line.

[0296] FIG38 is a diagram showing the invasion effect of strain SWT1004 on different cancer cell lines, wherein BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0297] (A) Effect on invasion of CT26 cell line;

[0298] (B) Effect on invasion of EMT6 cell line;

[0299] (C) Invasion effect on MB49 cell line;

[0300] (D) Invasion effect on RM-1 cell line;

[0301] (E) Invasion effect on A549 cell line;

[0302] (F) Invasion effect on K7M2 cell line;

[0303] (G) Invasion effect on B16F10 cell line;

[0304] (H) Invasion effect on MFC cell lines;

[0305] (I) Invasion effect on SCC7 cell line;

[0306] (J) Invasion effect on Hepa1-6 cell line;

[0307] (K) Invasion effect on Pan02 cell line;

[0308] (L) Invasion effect on ID8 cell line.

[0309] Figure 39 supplements Figure 38 , which shows the invasion effect of strain SWT1004 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0310] (M) Invasion effect on Renca cell line;

[0311] (N) Invasion effect on Neuro-2a cell line.

[0312] Figure 40 shows the invasion effect of strain SWT1005 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0313] (A) Effect on invasion of CT26 cell line;

[0314] (B) Effect on invasion of EMT6 cell line;

[0315] (C) Invasion effect on MB49 cell line;

[0316] (D) Invasion effect on RM-1 cell line;

[0317] (E) Invasion effect on A549 cell line;

[0318] (F) Invasion effect on K7M2 cell line;

[0319] (G) Invasion effect on B16F10 cell line;

[0320] (H) Invasion effect on MFC cell lines;

[0321] (I) Invasion effect on SCC7 cell line;

[0322] (J) Invasion effect on Hepa1-6 cell line;

[0323] (K) Invasion effect on Pan02 cell line;

[0324] (L) Invasion effect on ID8 cell line.

[0325] Figure 41 supplements Figure 40 , showing the invasion effect of strain SWT1005 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0326] (M) Invasion effect on Renca cell line;

[0327] (N) Invasion effect on Neuro-2a cell line.

[0328] Figure 42 shows the invasion effect of strain SWT1009 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0329] (A) Effect on invasion of CT26 cell line;

[0330] (B) Effect on invasion of EMT6 cell line;

[0331] (C) Invasion effect on MB49 cell line;

[0332] (D) Invasion effect on RM-1 cell line;

[0333] (E) Invasion effect on A549 cell line;

[0334] (F) Invasion effect on K7M2 cell line;

[0335] (G) Invasion effect on B16F10 cell line;

[0336] (H) Invasion effect on MFC cell lines;

[0337] (I) Invasion effect on SCC7 cell line;

[0338] (J) Invasion effect on Hepa1-6 cell line;

[0339] (K) Invasion effect on Pan02 cell line;

[0340] (L) Invasion effect on ID8 cell line.

[0341] Figure 43 supplements Figure 42 , showing the invasion effect of strain SWT1009 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0342] (M) Invasion effect on Renca cell line;

[0343] (N) Invasion effect on Neuro-2a cell line.

[0344] Figure 44 shows the invasion effect of strain SWT1010 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0345] (A) Effect on invasion of CT26 cell line;

[0346] (B) Effect on invasion of EMT6 cell line;

[0347] (C) Invasion effect on MB49 cell line;

[0348] (D) Invasion effect on RM-1 cell line;

[0349] (E) Invasion effect on A549 cell line;

[0350] (F) Invasion effect on K7M2 cell line;

[0351] (G) Invasion effect on B16F10 cell line;

[0352] (H) Invasion effect on MFC cell lines;

[0353] (I) Invasion effect on SCC7 cell line;

[0354] (J) Invasion effect on Hepa1-6 cell line;

[0355] (K) Invasion effect on Pan02 cell line;

[0356] (L) Invasion effect on ID8 cell line.

[0357] Figure 45 supplements Figure 44 , showing the invasion effect of strain SWT1010 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0358] (M) Invasion effect on Renca cell line;

[0359] (N) Invasion effect on Neuro-2a cell line.

[0360] Figure 46 shows the invasion effect of strain SWT1012 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0361] (A) Effect on invasion of CT26 cell line;

[0362] (B) Effect on invasion of EMT6 cell line;

[0363] (C) Invasion effect on MB49 cell line;

[0364] (D) Invasion effect on RM-1 cell line;

[0365] (E) Invasion effect on A549 cell line;

[0366] (F) Invasion effect on K7M2 cell line;

[0367] (G) Invasion effect on B16F10 cell line;

[0368] (H) Invasion effect on MFC cell lines;

[0369] (I) Invasion effect on SCC7 cell line;

[0370] (J) Invasion effect on Hepa1-6 cell line;

[0371] (K) Invasion effect on Pan02 cell line;

[0372] (L) Invasion effect on ID8 cell line.

[0373] Figure 47 supplements Figure 46 , which shows the invasion effect of strain SWT1012 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0374] (M) Invasion effect on Renca cell line;

[0375] (N) Invasion effect on Neuro-2a cell line.

[0376] Figure 48 shows the invasion effect of strain SWT1013 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0377] (A) Effect on invasion of CT26 cell line;

[0378] (B) Effect on invasion of EMT6 cell line;

[0379] (C) Invasion effect on MB49 cell line;

[0380] (D) Invasion effect on RM-1 cell line;

[0381] (E) Invasion effect on A549 cell line;

[0382] (F) Invasion effect on K7M2 cell line;

[0383] (G) Invasion effect on B16F10 cell line;

[0384] (H) Invasion effect on MFC cell lines;

[0385] (I) Invasion effect on SCC7 cell line;

[0386] (J) Invasion effect on Hepa1-6 cell line;

[0387] (K) Invasion effect on Pan02 cell line;

[0388] (L) Invasion effect on ID8 cell line.

[0389] Figure 49 supplements Figure 48 , showing the invasion effect of strain SWT1013 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0390] (M) Invasion effect on Renca cell line;

[0391] (N) Invasion effect on Neuro-2a cell line.

[0392] Figure 50 shows the invasion effect of strain SWT1021 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0393] (A) Effect on invasion of CT26 cell line;

[0394] (B) Effect on invasion of EMT6 cell line;

[0395] (C) Invasion effect on MB49 cell line;

[0396] (D) Invasion effect on RM-1 cell line;

[0397] (E) Invasion effect on A549 cell line;

[0398] (F) Invasion effect on K7M2 cell line;

[0399] (G) Invasion effect on B16F10 cell line;

[0400] (H) Invasion effect on MFC cell lines;

[0401] (I) Invasion effect on SCC7 cell line;

[0402] (J) Invasion effect on Hepa1-6 cell line;

[0403] (K) Invasion effect on Pan02 cell line;

[0404] (L) Invasion effect on ID8 cell line.

[0405] Figure 51 supplements Figure 50, showing the invasion effect of strain SWT1021 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0406] (M) Invasion effect on Renca cell line;

[0407] (N) Invasion effect on Neuro-2a cell line.

[0408] Figure 52 shows the invasion effect of strain SWT1025 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0409] (A) Effect on invasion of CT26 cell line;

[0410] (B) Effect on invasion of EMT6 cell line;

[0411] (C) Invasion effect on MB49 cell line;

[0412] (D) Invasion effect on RM-1 cell line;

[0413] (E) Invasion effect on A549 cell line;

[0414] (F) Invasion effect on K7M2 cell line;

[0415] (G) Invasion effect on B16F10 cell line;

[0416] (H) Invasion effect on MFC cell lines;

[0417] (I) Invasion effect on SCC7 cell line;

[0418] (J) Invasion effect on Hepa1-6 cell line;

[0419] (K) Invasion effect on Pan02 cell line;

[0420] (L) Invasion effect on ID8 cell line.

[0421] Figure 53 supplements Figure 52 and shows the invasion effect of strain SWT1025 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0422] (M) Invasion effect on Renca cell line;

[0423] (N) Invasion effect on Neuro-2a cell line.

[0424] Figure 54 shows the invasion effect of strain SWT1033 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0425] (A) Effect on invasion of CT26 cell line;

[0426] (B) Effect on invasion of EMT6 cell line;

[0427] (C) Invasion effect on MB49 cell line;

[0428] (D) Invasion effect on RM-1 cell line;

[0429] (E) Invasion effect on A549 cell line;

[0430] (F) Invasion effect on K7M2 cell line;

[0431] (G) Invasion effect on B16F10 cell line;

[0432] (H) Invasion effect on MFC cell lines;

[0433] (I) Invasion effect on SCC7 cell line;

[0434] (J) Invasion effect on Hepa1-6 cell line;

[0435] (K) Invasion effect on Pan02 cell line;

[0436] (L) Invasion effect on ID8 cell line.

[0437] Figure 55 supplements Figure 54 , showing the invasion effect of strain SWT1033 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0438] (M) Invasion effect on Renca cell line;

[0439] (N) Invasion effect on Neuro-2a cell line.

[0440] Figure 56 shows the invasion effect of strain SWT1035 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0441] (A) Effect on invasion of CT26 cell line;

[0442] (B) Effect on invasion of EMT6 cell line;

[0443] (C) Invasion effect on MB49 cell line;

[0444] (D) Invasion effect on RM-1 cell line;

[0445] (E) Invasion effect on A549 cell line;

[0446] (F) Invasion effect on K7M2 cell line;

[0447] (G) Invasion effect on B16F10 cell line;

[0448] (H) Invasion effect on MFC cell lines;

[0449] (I) Invasion effect on SCC7 cell line;

[0450] (J) Invasion effect on Hepa1-6 cell line;

[0451] (K) Invasion effect on Pan02 cell line;

[0452] (L) Invasion effect on ID8 cell line.

[0453] Figure 57 supplements Figure 56 , showing the invasion effect of strain SWT1035 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0454] (M) Invasion effect on Renca cell line;

[0455] (N) Invasion effect on Neuro-2a cell line.

[0456] Figure 58 shows the invasion effect of strain SWT1036 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0457] (A) Effect on invasion of CT26 cell line;

[0458] (B) Effect on invasion of EMT6 cell line;

[0459] (C) Invasion effect on MB49 cell line;

[0460] (D) Invasion effect on RM-1 cell line;

[0461] (E) Invasion effect on A549 cell line;

[0462] (F) Invasion effect on K7M2 cell line;

[0463] (G) Invasion effect on B16F10 cell line;

[0464] (H) Invasion effect on MFC cell lines;

[0465] (I) Invasion effect on SCC7 cell line;

[0466] (J) Invasion effect on Hepa1-6 cell line;

[0467] (K) Invasion effect on Pan02 cell line;

[0468] (L) Invasion effect on ID8 cell line.

[0469] Figure 59 supplements Figure 58 and shows the invasion effect of strain SWT1036 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0470] (M) Invasion effect on Renca cell line;

[0471] (N) Invasion effect on Neuro-2a cell line.

[0472] Figure 60 shows the invasion effect of strain SWT1038 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0473] (A) Effect on invasion of CT26 cell line;

[0474] (B) Effect on invasion of EMT6 cell line;

[0475] (C) Invasion effect on MB49 cell line;

[0476] (D) Invasion effect on RM-1 cell line;

[0477] (E) Invasion effect on A549 cell line;

[0478] (F) Invasion effect on K7M2 cell line;

[0479] (G) Invasion effect on B16F10 cell line;

[0480] (H) Invasion effect on MFC cell lines;

[0481] (I) Invasion effect on SCC7 cell line;

[0482] (J) Invasion effect on Hepa1-6 cell line;

[0483] (K) Invasion effect on Pan02 cell line;

[0484] (L) Invasion effect on ID8 cell line.

[0485] Figure 61 supplements Figure 60, which shows the invasion effect of strain SWT1038 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0486] (M) Invasion effect on Renca cell line;

[0487] (N) Invasion effect on Neuro-2a cell line.

[0488] Figure 62 shows the invasion effect of strain SWT1063 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC are the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0489] (A) Effect on invasion of CT26 cell line;

[0490] (B) Effect on invasion of EMT6 cell line;

[0491] (C) Invasion effect on MB49 cell line;

[0492] (D) Invasion effect on RM-1 cell line;

[0493] (E) Invasion effect on A549 cell line;

[0494] (F) Invasion effect on K7M2 cell line;

[0495] (G) Invasion effect on B16F10 cell line;

[0496] (H) Invasion effect on MFC cell lines;

[0497] (I) Invasion effect on SCC7 cell line;

[0498] (J) Invasion effect on Hepa1-6 cell line;

[0499] (K) Invasion effect on Pan02 cell line;

[0500] (L) Invasion effect on ID8 cell line.

[0501] Figure 63 supplements Figure 62 and shows the invasion effect of strain SWT1063 on different cancer cell lines, where BF represents bright field, the bright spots shown in FITC represent the fluorescent signals of Salmonella after invasion, and Merge represents the merged image of BF and FITC.

[0502] (M) Invasion effect on Renca cell line;

[0503] (N) Invasion effect on Neuro-2a cell line.

[0504] Figure 64 compares the killing effects of strain SWT005, strain SWT1001 and strain SWT1002 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7 and MB49 cells.

[0505] (A) Comparison of the killing effects of strains SWT005 and SWT1001 on different cancer cell lines;

[0506] (B) Comparison of the killing effects of strains SWT005 and SWT1002 on different cancer cell lines.

[0507] Figure 65 compares the killing effects of strain SWT005, strain SWT1004 and strain SWT1005 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7 and MB49 cells.

[0508] (A) Comparison of the killing effects of strains SWT005 and SWT1004 on different cancer cell lines;

[0509] (B) Comparison of the killing effects of strains SWT005 and SWT1005 on different cancer cell lines.

[0510] Figure 66 compares the killing effects of strain SWT005, strain SWT1009 and strain SWT1010 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7 and MB49 cells.

[0511] (A) Comparison of the killing effects of strains SWT005 and SWT1009 on different cancer cell lines;

[0512] (B) Comparison of the killing effects of strains SWT005 and SWT1010 on different cancer cell lines.

[0513] Figure 67 compares the killing effects of strain SWT005, strain SWT1012 and strain SWT1013 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7 and MB49 cells.

[0514] (A) Comparison of the killing effects of strains SWT005 and SWT1012 on different cancer cell lines;

[0515] (B) Comparison of the killing effects of strains SWT005 and SWT1013 on different cancer cell lines.

[0516] Figure 68 compares the killing effects of strain SWT005, strain SWT1021 and strain SWT1025 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7 and MB49 cells.

[0517] (A) Comparison of the killing effects of strains SWT005 and SWT1021 on different cancer cell lines;

[0518] (B) Comparison of the killing effects of strains SWT005 and SWT1025 on different cancer cell lines.

[0519] Figure 69 compares the killing effects of strain SWT005, strain SWT1033 and strain SWT1035 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7 and MB49 cells.

[0520] (A) Comparison of the killing effects of strains SWT005 and SWT1033 on different cancer cell lines;

[0521] (B) Comparison of the killing effects of strains SWT005 and SWT1035 on different cancer cell lines.

[0522] Figure 70 compares the killing effects of strain SWT005, strain SWT1036 and strain SWT1038 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7 and MB49 cells.

[0523] (A) Comparison of the killing effects of strain SWT005 and strain SWT1036 on different cancer cell lines;

[0524] (B) Comparison of the killing effects of strains SWT005 and SWT1038 on different cancer cell lines.

[0525] Figure 71 compares the killing effects of strain SWT005 and strain SWT1063 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, Renca, CT26, ID8, Neuro-2a, SCC7, and MB49 cells.

[0526] Figure 72 shows the targeting evaluation of strain SWT005 in a mouse tumor model.

[0527] Bacterial counts in the heart, liver, spleen, lung, kidney, and tumor of mice on day 1 (D1), day 5 (D5), and day 11 (D11) after SWT005 was injected into the tail vein.

[0528] Figure 73 shows the targeting evaluation of strains SWT1001, SWT1002, SWT1004, and SWT1005 in mouse tumor models.

[0529] Bacterial counts in the heart, liver, spleen, lung, kidney, and tumor of mice were measured on the 1st day (D1), 5th day (D5), and 11th day (D11) after the mice were injected with Salmonella via the tail vein. Statistical analysis was performed by comparing with the strain SWT005.

[0530] (A) Strain SWT1001;

[0531] (B) Strain SWT1002;

[0532] (C) Strain SWT1004;

[0533] (D) Strain SWT1005.

[0534] Figure 74 shows the targeting evaluation of strains SWT1009, SWT1010, SWT1012, and SWT1013 in mouse tumor models.

[0535] Bacterial counts in the heart, liver, spleen, lung, kidney, and tumor of mice were measured on the 1st day (D1), 5th day (D5), and 11th day (D11) after the mice were injected with Salmonella via the tail vein. Statistical analysis was performed by comparing with the strain SWT005.

[0536] (A) Strain SWT1009;

[0537] (B) Strain SWT1010;

[0538] (C) Strain SWT1012;

[0539] (D) Strain SWT1013.

[0540] Figure 75 shows the targeting evaluation of strains SWT1021, SWT1025, SWT1033, and SWT1035 in mouse tumor models.

[0541] Bacterial counts in the heart, liver, spleen, lung, kidney, and tumor of mice were measured on the 1st day (D1), 5th day (D5), and 11th day (D11) after the mice were injected with Salmonella via the tail vein. Statistical analysis was performed by comparing with the strain SWT005.

[0542] (A) Strain SWT1021;

[0543] (B) strain SWT1025;

[0544] (C) Strain SWT1033;

[0545] (D) Strain SWT1035.

[0546] Figure 76 shows the targeting evaluation of strains SWT1036, SWT1038, and SWT1063 in mouse tumor models.

[0547] Bacterial counts in the heart, liver, spleen, lung, kidney, and tumor of mice were measured on the 1st day (D1), 5th day (D5), and 11th day (D11) after the mice were injected with Salmonella via the tail vein. Statistical analysis was performed by comparing with the strain SWT005.

[0548] (A) strain SWT1036;

[0549] (B) strain SWT1038;

[0550] (C) Strain SWT1063.

[0551] Figure 77 Comparison of the tumor inhibitory effects of strain SWT005, strain SWT1001, strain SWT1002, strain SWT1004, and strain SWT1005 in a mouse tumor model, with Vehicle serving as the control group.

[0552] (A) Comparison of the tumor inhibitory effects of strains SWT005 and SWT1001 in mouse tumor models;

[0553] (B) Comparison of the tumor inhibitory effects of strains SWT005 and SWT1002 in mouse tumor models;

[0554] (C) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1004 in mouse tumor models;

[0555] (D) Comparison of the tumor inhibitory effects of strains SWT005 and SWT1005 in mouse tumor models.

[0556] Figure 78 compares the tumor inhibitory effects of strain SWT005 with strains SWT1009, SWT1010, SWT1012, and SWT1013 in a mouse tumor model, with Vehicle serving as the control group.

[0557] (A) Comparison of the tumor inhibitory effects of strains SWT005 and SWT1009 in mouse tumor models;

[0558] (B) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1010 in mouse tumor models;

[0559] (C) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1012 in mouse tumor models;

[0560] (D) Comparison of the tumor inhibitory effects of strains SWT005 and SWT1013 in mouse tumor models.

[0561] FIG79 compares the tumor inhibitory effects of strain SWT005, strain SWT1021, strain SWT1025, strain SWT1033, and strain SWT1035 in a mouse tumor model, with Vehicle serving as the control group.

[0562] (A) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1021 in mouse tumor models;

[0563] (B) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1025 in mouse tumor models;

[0564] (C) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1033 in mouse tumor models;

[0565] (D) Comparison of the tumor inhibitory effects of strains SWT005 and SWT1035 in mouse tumor models.

[0566] Figure 80 Comparison of the tumor inhibitory effects of strain SWT005, strain SWT1036, strain SWT1038, and strain SWT1063 in a mouse tumor model, with Vehicle serving as the control group.

[0567] (A) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1036 in mouse tumor models;

[0568] (B) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1038 in mouse tumor models;

[0569] (C) Comparison of the tumor inhibitory effects of strains SWT005 and SWT1063 in mouse tumor models.

[0570] FIG81 is a diagram showing the verification of the PCR amplification product of SWT007 identified by agarose gel electrophoresis.

[0571] Figure 82 is a diagram of the strain verification library after the insertion of the hypoxia-specific gene expression cassette (dadX is an essential gene) identified by agarose gel electrophoresis.

[0572] (A)SWT2001, SWT2002, SWT2004, SWT2005;

[0573] (B)SWT2009, SWT2010, SWT2012, SWT2013;

[0574] (C)SWT2033, SWT2035, SWT2036, SWT2038;

[0575] (D) SWT2021, SWT2025;

[0576] (E)SWT2063.

[0577] Figure 83 shows the oxygen adaptability verification of strains SWT2001, SWT2002, SWT2004 and SWT2005.

[0578] (A) SWT2001 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0579] (B) SWT2002 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0580] (C) SWT2004 was cultured on LB plates without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0581] (D) SWT2005 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0582] Figure 84 shows the oxygen adaptability verification of strains SWT2009, SWT2010, SWT2012 and SWT2013.

[0583] (A) SWT2009 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0584] (B) SWT2010 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0585] (C) SWT2012 cells were cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0586] (D) SWT2013 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0587] Figure 85 shows the oxygen adaptability verification of strains SWT2033, SWT2035, SWT2036 and SWT2038.

[0588] (A) SWT2033 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0589] (B) SWT2035 was cultured on LB plates without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0590] (C) SWT2036 was cultured on LB plates without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0591] (D) SWT2038 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0592] Figure 86 shows the oxygen adaptability verification of strains SWT2021, SWT2025 and SWT2063.

[0593] (A) SWT2021 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0594] (B) SWT2025 was cultured on LB plates without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0595] (C) SWT2063 was cultured on LB plates without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0596] Figure 87 shows the oxygen adaptability verification of strains SWT1065, SWT1066, SWT1068 and SWT1069.

[0597] (A) SWT1065 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0598] (B) SWT1066 was cultured on LB plates without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0599] (C) SWT1068 cells were cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0600] (D) SWT1069 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0601] Figure 88 shows the oxygen adaptability verification of strains SWT1041, SWT1042, SWT1044 and SWT1045.

[0602] (A) SWT1041 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0603] (B) SWT1042 cells were cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0604] (C) SWT1044 was cultured on LB plates without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0605] (D) SWT1045 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0606] Figure 89 shows the oxygen adaptability verification of strains SWT1008, SWT1016 and SWT1024.

[0607] (A) SWT1008 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0608] (B) SWT1016 was cultured on LB plates without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0609] (C) SWT1024 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0610] Figure 90 Verification of oxygen adaptability of strains SWT1073, SWT1074 and SWT1075.

[0611] (A) SWT1073 was cultured on LB plates without D-alanine under anaerobic conditions.

[0612] The following is aerobic culture;

[0613] (B) SWT1074 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic conditions and the lower plate showing aerobic conditions.

[0614] (C) SWT1075 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions. DETAILED DESCRIPTION

[0615] The present application will be further described in detail below with reference to the accompanying drawings and examples, through which the features and advantages of the present application will become more clearly understood.

[0616] Example 1: Construction of a hypoxia-specific gene expression cassette with the alr gene as an essential gene for survival

[0617] In this embodiment, the forward hypoxia promoters are 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), the survival essential gene is the alr gene of Salmonella (SEQ ID No. 17), and the reverse hyperoxia promoters are 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 to construct a hypoxia-specific gene expression cassette.

[0618] The combination of hypoxia-specific gene expression cassettes is shown in Table 1.

[0619] Table 1 Combinations of forward hypoxia promoters and reverse hyperxia promoters (where the essential survival gene is the alr gene of Salmonella)

[0620] (I) Construction of a positive hypoxia promoter clone library

[0621] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Industrial Microbiological Culture Collection) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) and inoculate it into 5 ml LB liquid medium and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The OD 600 The reading is between 2-3;

[0622] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium and mix it into the corresponding primer and high-fidelity PCR amplification enzyme (purchased from TAKARA) system, and then put the mixture into the PCR amplification device.

[0623] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes;

[0624] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0625] 4. Mix the recovered product with the endonuclease (purchased from NEB) digestion system and incubate at 37°C for 1 hour;

[0626] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0627] The primers and endonucleases used for different forward hypoxia promoters are as follows:

[0628] 1. yhbU-S (SEQ ID No. 1): strain SWT001 was amplified using primers SWTO19 and 20, and the product was double-cut with NotI and HindIII and recovered;

[0629] 2. ynfK-E (SEQ ID No. 2): amplified from E. coli strain DH10B using primers SWTO21, 22, and the product was double-cleaved with NotI and HindIII and recovered;

[0630] 3. tdcA-S (SEQ ID No. 3): strain SWT001 was amplified using primers SWTO23, 24, and the product was double-cut with NotI and HindIII and recovered;

[0631] 4. tdcA-E (SEQ ID No. 4): amplified from E. coli strain DH10B using primers SWTO25, 26, and the product was double-cleaved with NotI and HindIII and recovered;

[0632] 5. yecH-E (SEQ ID No. 5): amplified from E. coli strain DH10B using primers SWTO27, 28, and the product was double-cleaved with NotI and HindIII and recovered;

[0633] 6. ynfK-S (SEQ ID No. 6): strain SWT001 was amplified using primers SWTO29, 30, and the product was double-cut with NotI and HindIII and recovered;

[0634] 7. cydA-E (SEQ ID No. 7): amplified from E. coli strain DH10B using primers SWTO31, 32, and the product was double-cleaved with NotI and HindIII and recovered;

[0635] 8. focA-E (SEQ ID No. 8): amplified from E. coli strain DH10B using primers SWTO33, 34, and the product was double-cleaved with NotI and HindIII and recovered.

[0636] 9. yhbU-E (SEQ ID No. 48): The E. coli strain DH10B was amplified using primers SWTO82, 83. The product was double-cleaved with NotI and HindIII and recovered.

[0637] The results of verification of the forward hypoxia promoter clone library by agarose gel electrophoresis are shown in Figures 1(A) and 1(B).

[0638] (2) Construction of a gene library essential for survival

[0639] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Industrial Microbiological Culture Collection) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) and inoculate it into 5 ml of LB liquid medium and incubate it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0640] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium and mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then place the mixture into the PCR amplification device.

[0641] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes;

[0642] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0643] 4. Mix the recovered product with the enzyme digestion system and incubate at 37 degrees for 1 hour;

[0644] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0645] The essential genes for survival from different sources are as follows:

[0646] 1. Salmonella alr gene (SEQ ID No. 17): strain SWT001 was amplified using primers SWTO35 and 36, and the product was double-cut with HindIII and XhoI and recovered;

[0647] 2. Escherichia coli alr gene (SEQ ID No. 21): strain DH10B was amplified using primers SWTO37 and 38, and the product was double-cut with HindIII and XhoI and recovered;

[0648] 3. Salmonella dadX gene (SEQ ID No. 19): strain SWT001 was amplified using primers SWTO39 and 40, and the product was double-cut with HindIII and XhoI and recovered;

[0649] 4. E. coli dadX gene (SEQ ID No. 23): strain DH10B was amplified using primers SWTO41 and 42, and the product was double-cut with HindIII and XhoI and recovered.

[0650] The results of verification of the essential survival gene library by agarose gel electrophoresis are shown in FIG2 .

[0651] (III) Construction of the reverse hyperoxia promoter library

[0652] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC, China Industrial Microbiological Culture Collection) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) and inoculate it into 5 ml of LB liquid medium. Place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0653] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium and mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then place the mixture into the PCR amplification device.

[0654] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; 4°C for 5 minutes;

[0655] 3. The amplified PCR product is purified and recovered through a DNA gel recovery system;

[0656] 4. Mix the recovered product with the enzyme digestion system and incubate at 37°C for 1 hour;

[0657] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0658] The primers and endonucleases used for different reverse hyperoxia promoters are as follows:

[0659] 1. cyoA-S (SEQ ID No. 9): strain SWT001 was amplified using primers SWTO43 and 44, and the product was double-cleaved with XhoI and PstI and recovered;

[0660] 2. phoH-S (SEQ ID No. 10): strain SWT001 was amplified using primers SWTO45 and 46, and the product was double-cut with XhoI and PstI and recovered;

[0661] 3. lldP-S (SEQ ID No. 11): strain SWT001 was amplified using primers SWTO47 and 48, and the product was double-cut with XhoI and PstI and recovered;

[0662] 4. argT-S (SEQ ID No. 12): strain SWT001 was amplified using primers SWTO49 and 50, and the product was double-cut with XhoI and PstI and recovered;

[0663] 5. ydcI-S (SEQ ID No. 13): strain SWT001 was amplified using primers SWTO51 and 52, and the product was double-cut with XhoI and PstI and recovered;

[0664] 6. phoH-E (SEQ ID No. 14): strain DH10B was amplified using primers SWTO53 and 54, and the product was double-cut with XhoI and PstI and recovered;

[0665] 7. lldP-E (SEQ ID No. 15): strain DH10B was amplified using primers SWTO55 and 56, and the product was double-cut with XhoI and PstI and recovered;

[0666] 8. ydcI-E (SEQ ID No. 16): strain DH10B was amplified using primers SWTO57 and 58, and the product was double-cut with XhoI and PstI and recovered.

[0667] 9. mqo (SEQ ID No. 62): strain DH10B was amplified using primers SWTO7 and 8, and the product was double-cut with XhoI and PstI and recovered.

[0668] The results of verification of the reverse hyperoxia promoter clone library by agarose gel electrophoresis are shown in Figures 3(A) and 3(B).

[0669] (IV) Construction of a combinatorial library of forward hypoxia promoters, essential survival genes, and reverse hyperoxia promoters

[0670] The composition of the hypoxia-specific gene expression cassette is shown in FIG4 , which is composed of a forward hypoxia promoter, a survival-essential gene, and a reverse hyperoxia promoter in sequence.

[0671] As shown in Table 1, a cloned library was formed by combining sequences of the forward hypoxia promoter library digested with NotI and HindIII, the survival essential gene Salmonella alr gene (SEQ ID No. 17) digested with HindIII and XhoI, and the reverse hyperoxia promoter library digested with XhoI and PstI, and ligated into the plasmid pSWT003 vector (vector backbone, pBlueScript SK(+), purchased from Biowind) digested with SpeI and PstI, and the plasmid pSWT007 vector (containing double-sided same-direction loxP sequences and a DNA fragment of the chloramphenicol resistance gene (SEQ ID No. 61)) digested with SpeI and NotI.

[0672] The products amplified from Salmonella SWT001 or Escherichia coli DH10B using the above primers and recovered by enzyme digestion were combined and ligated by enzyme ligation reaction, and coated on LB plates containing 25 μg / ml chloramphenicol to obtain the corresponding plasmids shown in Table 1.

[0673] The construction of each strain of the present invention is as follows:

[0674] 1. Wild-type Salmonella Typhimurium (SWT001)

[0675] Wild-type Salmonella typhimurium (SWT001) was purchased from China Industrial Microbiology Culture Collection Center (CICC).

[0676] 2. Construction of Salmonella (SWT002) containing temperature-inducible lambda-RED recombinase and loxp-CRE enzyme system

[0677] The lambda-RED recombination system is widely used for homologous recombination in Gram-negative bacteria. In the present invention, this system is comprised of the plasmid pSWT001. As shown in Figure 5 , plasmid pSWT001 contains the lambda-RED recombinase module (SEQ ID No. 60) (functionally similar to the pSIM6 plasmid vector from Biowind) and the loxP-Cre recombinase module (SEQ ID No. 60) (functionally similar to the 705-Cre plasmid vector from Gene Bridges). The lambda-RED recombinase module consists of three recombinases: EXO, BET, and GAM. These recombinases are controlled by the CI857 temperature regulator. Therefore, expression is lost at 32°C and is only activated at temperatures above 37°C. Under temperature-induced conditions, the recombinase EXO cleaves the 5' end of double-stranded linear DNA, creating a single-stranded DNA protrusion at the 3' end. This single-stranded DNA is bound by the BET protein and protected from degradation by other nucleases. GAM inhibits endogenous bacterial nucleases. The homologous arms of homologous recombination are about 35-50bp and are added on both sides of the DNA fragment to be recombined through PCR. The advantage of this technology is that it can accurately target the target region of the bacterial chromosome without causing additional mutations. The outermost parts of the recombined double-stranded DNA fragments include the homologous arm sequences of the position where recombination is required, which contain loxp sequences in the same direction on both sides, totaling 34bp. The sites of the double loxp in the same direction contain the chloramphenicol resistance gene, which is used to screen recombinant bacteria. After successful recombination, the CRE enzyme system carried on the pSWT001 plasmid (also controlled by the CI857 temperature regulator) specifically recognizes the loxp sequence and can cut the sequence in the middle of the loxp in the same direction, leaving a loxp sequence, thereby eliminating the chloramphenicol resistance gene. Repeating the above operations can achieve continuous knockout and knock-in of genes.

[0678] The specific steps are as follows:

[0679] (1) Streak the strain SWT001 on an LB plate and culture it in a 37°C incubator overnight;

[0680] (2) Pick a single clone and inoculate it into 5 ml of LB liquid medium and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0681] (3) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3 Place on ice and let stand for 1 hour;

[0682] (4) Wash the cells three times with sterilized purified water;

[0683] (5) The recovered cells were mixed with 10 ng of plasmid pSWT001 and electroporated at a voltage of 1.8 kV;

[0684] (6) The electroporated cells were spread on LB plates containing 100 μg / ml ampicillin sodium and cultured overnight in a 32°C constant temperature incubator until a single clone colony was grown, named SWT002.

[0685] 3. Construction of attenuated Salmonella with aroA gene knockout (SWT003)

[0686] (1) A single clone of SWT002 was picked and inoculated into 5 ml of LB liquid culture medium containing 100 μg / ml ampicillin sodium, and placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0687] (2) Inoculate the culture into fresh LB liquid medium containing 100 μg / ml ampicillin sodium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0688] (3) Wash the cells three times with sterilized purified water.

[0689] (4) Prepare PCR products of SWTO1 and SWTO2.

[0690] Primers SWTO1 and SWTO2 were mixed with plasmid pSWT002 (containing 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 and placed in a PCR amplification device.

[0691] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0692] The amplified PCR products were subjected to agarose gel electrophoresis to verify the PCR amplification products of SWTO1 and SWTO2 of the aroA gene knockout fragments. The verification results are shown in Figure 6A.

[0693] The PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were measured using nanodrop.

[0694] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO1 and SWTO2 and electroporated at a voltage of 1.8 kV.

[0695] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0696] (7) Positive clones were identified by colony PCR, and the insertion of chloramphenicol resistance gene was identified using SWTO4, SWTO6, SWTO3 and SWTO5.

[0697] The PCR amplification product of the target fragment of strain SWT003 was verified by agarose gel electrophoresis, and the verification results are shown in Figure 6B.

[0698] (8) The positive single clone was inoculated into 5 ml of LB medium and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0699] 4. Construction of alr gene knockout attenuated bacteria (SWT004)

[0700] (1) A single clone of SWT003 was selected and inoculated into 5 ml of LB liquid culture medium, and then placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0701] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0702] (3) Wash the cells three times with sterilized purified water.

[0703] (4) Preparation of PCR products of SWTO70 and SWTO71.

[0704] Primers SWTO70 and SWTO71 were mixed with plasmid pSWT002 and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0705] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0706] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0707] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO70 and SWTO71 and electroporated at a voltage of 1.8 kV.

[0708] The PCR amplification products of SWTO70 and SWTO71 of the alr gene knockout fragments were verified by agarose gel electrophoresis. The verification results are shown in Figure 7A.

[0709] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0710] (7) Positive clones were identified by colony PCR, and SWTO72, SWTO5, SWTO6, and SWTO73 were used to identify the insertion of the chloramphenicol resistance gene.

[0711] The PCR amplification product of the target fragment of strain SWT004 was verified by agarose gel electrophoresis, and the verification results are shown in Figure 7B (the primers used were SWTO72, SWTO5, SWTO6, and SWTO73).

[0712] (8) The positive single clone was inoculated into 5 ml of LB medium and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0713] 5. Construction of YB1-like Salmonella (SWT005)

[0714] In order to study a more preferred oxygen-regulated promoter combination, the present invention constructed the YB1 Salmonella strain described in Chinese Patent CN104471057B as a prior art strain for comparison with the strain constructed by the present invention.

[0715] The YB1-like Salmonella strain includes aroA gene deficiency and an oxygen regulation system constructed by the forward low-oxygen promoter pepT, the essential gene asd, and the reverse high-oxygen promoter sodA.

[0716] (1) Construction of plasmid pSWT004

[0717] ① Construction of the pSWT004 plasmid as shown in FIG8 , which carries the chloramphenicol resistance gene cm (SEQ ID No. 43) flanked by loxp sequences, the pepT promoter (SEQ ID No. 44), the asd gene (SEQ ID No. 45), and the reverse sodA promoter (SEQ ID No. 47).

[0718] ② Plasmid pSWT002 was digested with NotI and the 1130 bp fragment was recovered. Strain SWT001 was amplified using primers SWTO11 and SWTO12, and the product was double-digested with NotI and HindIII and recovered.

[0719] Strain SWT001 was amplified using primers SWTO13 and SWTO14, and the product was double-cut with XhoI and HindIII and recovered.

[0720] Primers SWTO15 and SWTO16 were directly annealed to generate XhoI and PstI nicks, and then recovered.

[0721] ③ The plasmid pSWT003 (plasmid backbone) was double-cut with NotI and PstI, and the five fragments were connected by T4 DNA ligase. The plates were coated with ampicillin and chloramphenicol double-resistant LB plates, and single clones were screened and named pSWT004 (YB1-like construction plasmid, including cm-pepT-asd-sodA).

[0722] ④ Extract plasmid pSWT004, take 100 ng and mix with SacI and KpnI endonuclease system, incubate at 37°C for 1 hour, and identify by agarose gel electrophoresis, as shown in Figure 9.

[0723] (2) Construction of recombinant YB1-like Salmonella SWT005 (YB1-like)

[0724] Plasmid pSWT004 was amplified using primers SWTO17 and SWTO18, recovered, and recombined into SWT003 to construct YB1-like Salmonella SWT005.

[0725] ① Pick a single clone of SWT003 and inoculate it into 5 ml of LB liquid culture medium, and place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0726] ②Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue to culture for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0727] ③ Wash the bacteria 3 times with sterilized purified water and set aside.

[0728] ④ pSWT004 was amplified using primers SWTO17 and SWTO18, and the recovered PCR products were purified and recovered. The PCR amplification products of the cm-pepT-asd-sodA fragments SWTO17 and SWTO18 were verified by agarose gel electrophoresis. The verification results are shown in Figure 10. The DNA concentration and purity were determined by nanodrop.

[0729] ⑤ Mix the recovered bacteria with 100 ng of PCR product and electroporate at a voltage of 1.8 kV.

[0730] ⑥ Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml DAP (diaminopimelic acid), and culture them in a 32°C constant temperature incubator overnight until a single clone colony grows.

[0731] ⑦ Positive clones were identified by colony PCR. SWTO93, SWTO5, SWTO94, and SWTO95 were used to identify the insertion of the chloramphenicol resistance gene. The results of verification of the PCR amplification products of Salmonella SWT005 by agarose gel electrophoresis (SWTO93 and SWTO5, SWTO94 and SWTO95) are shown in Figure 11.

[0732] (3) Verification of oxygen adaptability of YB1-like Salmonella SWT005

[0733] ① Pick a single clone of YB1-like Salmonella SWT005 and inoculate it into 5 ml of LB liquid culture medium containing 100 μg / ml DAP (diaminopimelic acid), and place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0734] ② After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0735] ③ Calculate the volume of bacterial solution (μl) per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0736] ④ Take 10 μl of the above bacterial solution and spot it on two LB plates without DAP for culture, mark it as "1", and repeat three times, mark it as "a", "b", and "c".

[0737] ⑤ After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marked as "2".

[0738] ⑥ Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0739] ⑦ One of the plates was placed in an anaerobic jar at 37°C. The operation process was to place the anaerobic gas-generating bag (brand: Japan Mitsubishi, product number: D-119) in a 7.0L sealed culture jar (brand: Japan Mitsubishi, product number: D-112) and cultured in an anaerobic environment (oxygen concentration less than 0.01%) for 24 hours. The other plate was placed in an atmospheric environment at 37°C (21% oxygen concentration) and cultured for 24 hours.

[0740] The results of the oxygen adaptability verification test of YB1-like Salmonella SWT005 are shown in Figure 12.

[0741] From the oxygen adaptability verification data of YB1-like Salmonella SWT005, it can be concluded that YB1-like Salmonella SWT005 can grow normally in the absence of oxygen on the LB plate without DAP (as shown in Figure 12 (A)), and can grow normally in the presence of oxygen (atmospheric environment) at a concentration of 1 (10 -2 OD), concentration 2(10 -3 OD), concentration 3(10 -4 OD), concentration 4(10 -5 OD) showed obvious growth of the strains (as shown in Figure 12 (B)).

[0742] Therefore, it can be seen that YB1-like Salmonella SWT005 does not achieve strict regulation of strain growth in an aerobic environment, which is consistent with previous reports on YB1, further indicating that the oxygen regulation system of YB1 Salmonella SWT005 has certain defects.

[0743] Therefore, the present invention proposes an improved solution, which replaces the essential gene fragments on the one hand and replaces the front and back promoter fragments on the other hand, so as to achieve stricter regulation of bacteria in the presence of oxygen and be more inclined to target the hypoxic areas of tumors.

[0744] 6. Verification of essential gene replacement and construction of SWT006 strain

[0745] Based on the experimental data of YB1-like Salmonella SWT005, the present invention believes that the selection of the essential gene asd in YB1-like Salmonella SWT005 (pepT-asd-sodA strain) is not optimal.

[0746] Therefore, the present invention replaced the essential gene asd in the YB1-like Salmonella SWT005 oxygen regulation expression cassette with the alr gene, created a new hypoxia-specific regulatory expression cassette (pepT-alr-sodA), and verified its oxygen regulation function.

[0747] Since double mutations in the alr and dadX genes cause lethal traits, it is necessary to first construct the SWT004 strain (a strain with both the aroA and alr genes knocked out), and then integrate the hypoxia-specific regulatory expression cassette (pepT-alr-sodA) into the dadX gene site of the SWT004 strain (destroying its function) to construct the SWT006 strain.

[0748] (1) Construction of plasmid pSWT005

[0749] Based on SWT004, a strain with a pepT promoter, alr as an essential gene, and a sodA promoter in reverse was constructed by integrating a hypoxia-specific gene expression cassette into the dadX locus of SWT004. The construction process is as follows:

[0750] ① The plasmid pSWT004 (YB1-like plasmid, including cm-pepT-asd-sodA) was digested with HindIII and XhoI, the asd fragment was cut out, and the remaining fragments were recovered.

[0751] ② The strain SWT001 was amplified using primers SWTO35 and SWTO36 to obtain the amplified product of the alr gene of Salmonella. The product was double-cut with HindIII and XhoI and recovered.

[0752] ③ Connect the above fragments using T4 DNA ligase.

[0753] ④ Transform into DH10B competent cells, spread on LB plates containing ampicillin and chloramphenicol, and culture inverted at 37°C for 16 hours.

[0754] ⑤ Single clones were picked and identified by PCR using primers SWTO11 and SWTO36. The PCR amplification products of SWTO11 and SWTO36 were verified by agarose gel electrophoresis. The verification results are shown in Figure 13.

[0755] ⑥ Shake the bacteria and extract the plasmid to obtain plasmid pSWT005 (cm-pepT-alr-sodA element).

[0756] (2) Hypoxia-specific gene expression cassette pepT-alr-sodA strain (SWT006, aroA - &alr - Construction of pepT-alr-sodA

[0757] ① Pick a single clone of the alr gene-knockout attenuated bacteria SWT004 and inoculate it into 5 ml of LB liquid culture medium. Place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0758] ②Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue to culture for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0759] ③ Wash the bacteria 3 times with sterilized purified water and set aside.

[0760] ④ pSWT005 (cm-pepT-alr-sodA) was amplified using primers SWTO89 and SWTO90, and the purified PCR product was recovered. The concentration and purity of the recovered PCR product were determined by nanodrop.

[0761] ⑤ Mix the recovered bacteria with 100 ng of PCR product and electroporate at a voltage of 1.8 kV.

[0762] ⑥ Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and culture them in a 32°C constant temperature incubator overnight until a single clone colony grows.

[0763] ⑦ Positive clones were identified by colony PCR. The insertion of the chloramphenicol resistance gene was identified using SWTO87, SWTO76, SWTO59, and SWTO77. The PCR amplification product of Salmonella SWT006 was verified by agarose gel electrophoresis. The verification results are shown in Figure 14.

[0764] (3) Verification of oxygen adaptability of SWT006 strain

[0765] ① Pick a single clone of the SWT006 strain and inoculate it into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0766] ② After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0767] ③ Calculate the volume of bacterial solution per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0768] ④ Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, mark it as "1" and repeat three times, mark it as "a", "b", and "c".

[0769] ⑤ After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marked as "2".

[0770] ⑥ Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0771] ⑦ One of the plates 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 (21% oxygen concentration).

[0772] The results of the oxygen adaptability verification test of the SWT006 strain are shown in Figure 15(A). The SWT006 strain can grow normally in an anaerobic environment.

[0773] From the results of the oxygen adaptability verification of the SWT006 strain, it can be seen that, compared with the experimental results of the oxygen verification of the SWT005YB1-like, the alr gene was selected as the essential gene, which was better regulated than the asd gene of the SWT005YB1-like under aerobic conditions. As shown in Figure 15(B), only at a concentration of 1(10 -2 OD), concentration 2(10 -3 OD) and concentration 3(10 -4 OD) showed obvious strain growth.

[0774] In order to further optimize and achieve stricter oxygen regulation, the present invention further adjusts the forward hypoxia promoter and the reverse hyperoxia promoter of the hypoxia-specific gene expression cassette.

[0775] Example 2: Integration of the hypoxia-specific gene expression cassette into the dadX gene locus on the chromosome of the SWT004 strain (destroying its function)

[0776] (I) Construction of alr-deficient attenuated bacteria containing a hypoxia-specific gene expression cassette

[0777] 1. Pick a single clone of the alr gene-knockout attenuated bacteria SWT004 and inoculate it into 5 ml of fresh LB liquid culture medium. Place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0778] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue to culture for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0779] 3. Wash the bacteria 3 times with sterilized purified water.

[0780] 4. Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette. Mix primers SWTO78 and SWTO79 with the hypoxia-specific gene expression cassette plasmid and a high-fidelity PCR amplification enzyme system and place them in a PCR amplification device.

[0781] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 120 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0782] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0783] 5. Mix the recovered bacteria with 100 ng of PCR product and electroporate at a voltage of 1.8 kV.

[0784] 6. Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine and culture in a 32°C incubator overnight until a single clone colony grows.

[0785] 7. Inoculate the single clone into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours to allow the CRE enzyme to act and eliminate the chloramphenicol resistance gene.

[0786] 8. Using corresponding primers, clones were identified by colony PCR. The identification results are shown in Figure 16.

[0787] The information of the corresponding strains is shown in Table 2 , and the correspondence between the strains and the identification primers is shown in Table 3 .

[0788] Table 2 Strain construction table

[0789] Table 3 Correspondence between strains and identification primers

[0790] (II) Oxygen adaptability verification of the Salmonella strain library containing hypoxia-specific gene expression cassettes

[0791] 1. Pick a single clone of the strain containing the hypoxia-specific expression cassette and inoculate it into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine. Place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0792] 2. After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0793] 3. Calculate the volume of bacterial solution per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0794] 4. Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, mark it as "1" and repeat three times, mark it as "a", "b", and "c".

[0795] 5. After 10-fold serial dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0796] 6. Repeat this 10-fold gradient dilution until you reach the mark "8".

[0797] 7. One plate was cultured at 37°C in an anaerobic environment, and the other plate was cultured at 37°C in an atmospheric environment (21% oxygen concentration).

[0798] The results of the oxygen adaptability verification test of the strain containing the hypoxia-specific expression cassette are shown in Figures 17-31.

[0799] Based on the data of the aforementioned YB1-like (SWT005) and the essential gene replacement strain (SWT006), the present invention designed a new forward hypoxia promoter, essential genes, and reverse hyperoxia promoter, and formed a new hypoxia-specific gene expression cassette. The results showed that the Salmonella strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063 containing the hypoxia-specific gene expression cassette all showed better results than YB1-like (SWT005) and the essential gene replacement strain (SWT006) in the mutagenicity assessment.

[0800] (III) Oxygen concentration simulation of strain libraries containing hypoxia-specific gene expression cassettes

[0801] By using anaerobic gas production bags to consume the oxygen in the sealed culture tank, and using an oxygen meter to measure the oxygen concentration in the sealed culture tank, the oxygen concentration in the sealed culture tank can be fixed within a specific concentration range after using the anaerobic gas production bags for a certain period of time. The specific method is as follows:

[0802] The anaerobic gas production bag (brand: Japan Mitsubishi, product number: D-119) was placed in a 7.0 L sealed culture tank (brand: Japan Mitsubishi, product number: D-112) and an oxygen meter (brand: Meicheng Electrochemical, product number: OX-100A) was placed in the tank.

[0803] The above anaerobic gas production bag combination was used to verify the growth of the genetically modified strain under an oxygen concentration below 0.8%.

[0804] 1. Verification of the adaptability of the strain library containing hypoxia-specific gene expression cassettes to oxygen concentrations below 0.8%

[0805] Since the pathological hypoxic zone of a tumor is an oxygen concentration below 1%, the present invention intends to demonstrate that when the oxygen concentration is below 1% (or close to 1%), Salmonella transformed with the hypoxia-specific gene expression cassette can still grow normally.

[0806] Oxygen concentration control is accomplished by anaerobic gas production bags. After one hour of anaerobic gas production, the oxygen meter reading shows an oxygen concentration of 0.8-1%. Therefore, this experiment simulates the hypoxic environment within the tumor and verifies the growth of Salmonella on a petri dish after one hour of anaerobic gas production bags in a sealed culture jar. This also simulates the growth of Salmonella strains containing a hypoxia-specific gene expression cassette in the hypoxic environment within the tumor.

[0807] The operating procedures of the spot plate experiment are as follows:

[0808] (1) A single clone of the strain containing the hypoxia-specific expression cassette was selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0809] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0810] (3) Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0811] (4) Take 10 μl of the above bacterial solution and spot it on an LB plate containing D-alanine for culture, mark it as "1" and repeat three times, mark it as "a", "b", and "c".

[0812] (5) At the same time, take 10 μl of the above bacterial solution and spot it on another LB plate without D-alanine, mark it as "1", and spot three replicates, mark them as "a", "b", and "c".

[0813] (6) After 10-fold serial dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as “2”.

[0814] (7) Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0815] (8) The two plates were cultured at 37°C in a 0.8% oxygen environment.

[0816] As shown in Figure 32 , the present invention compared the growth of different strains (Figure 32 (A) SWT1001, Figure 32 (B) STW1005, Figure 32 (C) SWT1009, and Figure 32 (D) SWT1013) on LB plates without D-alanine to determine the growth performance of each strain at oxygen concentrations below 0.8%. Because the culture medium without D-alanine requires the corresponding Salmonella strains to rely on their own oxygen regulation systems for normal growth, the results confirmed that strains containing the hypoxia-specific expression cassette, which can grow normally in an anaerobic environment, can also grow normally at oxygen concentrations below 0.8%.

[0817] 2. Immunohistochemical staining of the strain containing the hypoxia-specific gene expression cassette (SWT1005) and the hypoxic area of ​​the tumor

[0818] To demonstrate the accumulation of Salmonella containing a hypoxia-specific gene expression cassette in the hypoxic areas of tumors, mouse tumor models injected with Salmonella through the tail vein were sectioned and stained with Salmonella and hypoxia-inducible factor HIF-1α antibodies to observe the distribution of Salmonella in the tumor model.

[0819] (1) Tumor model establishment

[0820] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, raised in an SPF environment) were subcutaneously inoculated with 1×10 6 CT26 mouse colon cells were used to establish a mouse colon cancer subcutaneous tumor model. 10-12 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS group and SWT1005 group, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0821] (2) Bacterial preparation

[0822] Taking strain SWT1005 as an example, streak the plate onto an LB plate supplemented with D-alanine and culture it statically in a 37°C incubator overnight. Pick a single colony of strain SWT1005 and inoculate it into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine. Incubate it in a shaker at 37°C, 220 rpm for 16 hours. Measure the absorbance (OD value) of the strain at 600 nm. Calculate and weigh the culture with an OD value of 1, wash it three times with PBS, and prepare a bacterial suspension in PBS.

[0823] (3) Tumor slice preparation

[0824] On the third day after administration, the animals were killed and dissected, and the tumors were removed and immersed in formalin fixative. Gradient dehydration was performed, and the tumors were embedded in paraffin. After complete cooling and solidification, the sections were sliced ​​and dewaxed. The sections were immersed in sodium citrate antigen retrieval solution, boiled for 10 minutes for antigen retrieval, and then rinsed with PBS three times. The sections after antigen retrieval were immersed in 3% hydrogen peroxide solution for 10 minutes, and then rinsed with PBS three times. After the catalase was removed, the sections were immersed in 2% BSA-PBS solution, blocked at room temperature for 1 hour, and then rinsed with PBS three times. Anti-Salmonella The primary antibody incubation solution of anti-HIF1α (purchased from abcam, product number ab35156) and anti-HIF1α (purchased from abcam, product number ab51608) was added dropwise to different blocked tissue sections, incubated at 4°C overnight, and then rinsed three times with PBS; the secondary antibody incubation solution was added dropwise to the tissue sections, incubated for 25 minutes, and then rinsed three times with PBS; DAB staining solution was added dropwise to the tissue sections, and counterstained with hematoxylin for 3 minutes; the tissue sections were dehydrated in sequence, and after dehydration, the sections were sealed with mounting media, and the sections were observed under a microscope and photographed, as shown in Figure 33.

[0825] The results showed that after antibody staining comparison, the brown (dark) area was antibody-positive, and Salmonella containing the hypoxia-specific gene expression box was found to be aggregated in the hypoxic area of ​​the tumor.

[0826] (IV) Evaluation of cell invasion of strains containing hypoxia-specific gene expression cassettes

[0827] pSWT006 (a prokaryotic promoter-controlled EGFP strong expression plasmid) was transferred into Salmonella modified with a hypoxia-specific gene expression cassette. The invasion of Salmonella into tumor cells was determined by observing the location of fluorescence and comparing the distribution of cells.

[0828] Strains containing hypoxia-specific gene expression cassettes were evaluated for their invasion effects on breast cancer (EMT6 cell line), osteosarcoma (K7M2 cell line), liver cancer (Hepa1-6 cell line), lung cancer (A549 cell line), melanoma (B16F10 cell line), kidney cancer (Renca cell line), gastric cancer (MFC cell line), pancreatic cancer (Pan02 cell line), prostate cancer (RM-1 cell line), colon cancer (CT26 cell line), ovarian cancer (ID8 cell line), neuroblastoma (Neuro-2a cell line), squamous cell carcinoma (SCC7 cell line), and bladder cancer (MB49 cell line).

[0829] 1. Evaluation of the effects of 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 on cancer cell invasion

[0830] (1) The strain modified with the hypoxia-specific gene expression cassette was transformed into the plasmid pSWT006 by electroporation to express EGFP protein, which facilitated the observation of cell invasion.

[0831] (2) The strain modified with the hypoxia-specific gene expression cassette carrying pSWT006 was streaked on an LB plate supplemented with D-alanine and cultured statically in a 37°C constant temperature incubator overnight.

[0832] (3) A single clone of the modified strain carrying pSWT006 was selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and placed in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0833] (4) Take 1OD of the culture and wash it three times with PBS.

[0834] (5) Add 2.5×10 6 The corresponding cancer cells were cultured in complete culture medium (DMEM basal medium + 10% fetal bovine serum + double antibody) in a 5% CO2 carbon dioxide incubator at 37°C overnight.

[0835] After counting cells from two wells, strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063 carrying the pSWT006-modified hypoxia-specific gene expression cassette were added according to the cell number at an infection ratio of 200:1. The cells were placed in an anaerobic jar at a concentration below 0.8% oxygen and incubated for 2 hours to allow bacterial invasion. The cells were then carefully washed with an equal volume of PBS, the supernatant aspirated, and DMEM containing 100 μg / mL gentamicin was added. The cells were incubated in an anaerobic jar at a concentration below 0.8% oxygen for another 24 hours, and images were captured under a fluorescence microscope. The invasion effects of the strain modified with the hypoxia-specific gene expression cassette on different cancer cell lines are shown in Figures 34-63.

[0836] From the above results, it can be seen that Salmonella containing the hypoxia-specific gene expression cassette has a strong ability to invade cells of various cancer cell lines. The growth and distribution of various Salmonella containing the hypoxia-specific gene expression cassette in cells can be clearly observed, indicating that Salmonella has universal invasion of cancer cells.

[0837] 2. The killing effect of strains containing hypoxia-specific gene expression cassettes on cancer cells under hypoxic conditions

[0838] By co-culturing with cancer cells under hypoxic conditions in vitro, 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 containing a hypoxia-specific gene expression cassette of the present invention on various cancer cells were verified.

[0839] (1) Streak the strain SWT005 on an LB plate supplemented with DAP; streak the modified strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063 on an LB plate supplemented with D-alanine, and culture them statically in a 37°C incubator overnight.

[0840] (2) Pick a single clone of the strain SWT005 and inoculate it into 5 ml of fresh LB liquid culture medium containing 100 μg / ml DAP; pick the modified strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038 and SWT1063 single clones and inoculate them into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and place them in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0841] (3) Measure the absorbance of the strain at 600 nm, take the culture with an OD value of 1, wash it twice with PBS, and then wash it once with the corresponding cell culture medium, and then resuspend the bacteria in the cell culture medium (infection ratio is 200:1).

[0842] (4) The above bacteria were 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 below 0.8% for 2 hours. The cells were then washed three times with PBS containing gentamicin, and the medium was changed and the cells were cultured in an anaerobic jar at an oxygen concentration below 0.8% for 24 hours.

[0843] (5) Mix the CCK8 reagent with the above cell suspension and incubate for 1 hour.

[0844] (6) The absorbance was measured using an enzyme-labeled instrument (detection wavelength: 450 nm) and the percentage of cytotoxicity was calculated.

[0845] Compared with the bacterium SWT005, the killing effects of strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038 and SWT1063 containing the 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 are shown in Figures 64-71.

[0846] The experimental results were analyzed using T-test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0847] The above results show that the Salmonella strains modified with the hypoxia-specific gene expression cassette have a significant killing effect on various cancer cells, and their effect is stronger than that of the strain SWT005 (YB1-like), indicating that the Salmonella modified with the hypoxia-specific gene expression cassette has universal killing effect on cancer cells.

[0848] 3. Targeting evaluation of strains containing hypoxia-specific gene expression cassettes in mouse tumor models

[0849] To explore the tumor targeting ability of strains containing hypoxia-specific gene expression cassettes in mouse tumor models, an EMT6 tumor mouse model was established.

[0850] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 EMT6 cancer cells were used to establish a mouse mammary subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 500 mm 3 Around 3:00 am, conduct the experiment.

[0851] The tumor-bearing mice were divided into 17 groups, including PBS (Vehicle), SWT005, SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038, and SWT1063, with 6 mice in each group. The tumor-bearing mice in the last 16 groups were injected with 1×10 7 Bacterial CFU counts were performed. Tumors were euthanized on day 1 (D1), day 5 (D5), and day 11 (D11) after administration, and the distribution of the aforementioned strains in normal tissues and organs, including the heart, liver, spleen, lungs, and kidneys, as well as in tumors, was assessed. Results show that the targeting of each Salmonella strain containing the hypoxia-specific gene expression cassette in a mouse tumor model, compared to the YB1-like strain, is evaluated as shown in Figures 72-76.

[0852] The above results show that the distribution of Salmonella strains containing hypoxia-specific gene expression cassettes in the heart, liver, spleen, lungs, and kidneys of the mouse model is less than that of YB1-like strains at all time points (no columns indicate that Salmonella was completely eliminated), but they can accumulate rapidly in tumors, which is significantly higher than that of YB1-like strains.

[0853] The experimental results were analyzed using T-test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0854] 4. Evaluation of Tumor Suppression of Strains Containing Hypoxia-Specific Gene Expression Cassettes in Mouse Tumor Models

[0855] To verify the inhibitory effect of strains containing hypoxia-specific gene expression cassettes on mouse tumor growth, the present invention designed an inhibitory test in the mouse EMT6 tumor model using the YB1-like strain SWT005 and the Salmonella strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038 and SWT1063 containing hypoxia-specific gene expression cassettes.

[0856] Establishment of EMT6 tumor mouse model: BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 EMT6 cancer cells were used to establish a mouse mammary subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 500 mm 3 Around 3:00 am, conduct the experiment.

[0857] (1) The strain SWT005 was streaked on an LB plate supplemented with DAP, and the constructed Salmonella strain was streaked on an LB plate supplemented with D-alanine, and then statically cultured in a 37°C constant temperature incubator overnight.

[0858] (2) A single clone of the strain SWT005 was selected and inoculated into 5 ml of LB liquid medium supplemented with DAP. A single clone of the constructed Salmonella strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038 and SWT1063 was selected and inoculated into 5 ml of LB liquid medium supplemented with D-alanine, and placed in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0859] (3) Measure the absorbance of the strain at 600 nm, take the culture with an OD value of 1, and wash it three times with PBS.

[0860] (4) 1×10 7 CFU counts of bacteria were administered to EMT6 model mice.

[0861] (5) The day of administration was designated as day 0. The length and width of the mouse tumor were measured on days 1, 3, 5, 7, 9, and 11 of administration, and the mouse tumor volume was calculated according to the following formula: Mouse tumor volume = length × width 2 ×0.52

[0862] The tumor inhibitory effects of each Salmonella strain containing the hypoxia-specific gene expression cassette in a mouse tumor model compared with the YB1-like strain SWT005 are shown in Figures 77-80.

[0863] The experimental results were analyzed using T-test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0864] Conclusion: The Salmonella strain containing the hypoxia-specific gene expression cassette has a significant inhibitory effect on tumor growth, which is better than the YB1-like strain SWT005.

[0865] Example 3: Construction of a hypoxia-specific gene expression cassette with dadX gene as an essential gene for survival

[0866] Since alanine racemase is expressed in two isoenzyme genes in the genomes of bacteria such as Salmonella and Escherichia coli, namely the alr gene and the dadX gene, the hypoxia-specific gene expression cassette was tested. To verify the efficacy of the hypoxia-specific gene expression cassette, the alr gene was replaced with the corresponding homologous dadX gene, thereby evaluating its oxygen regulation.

[0867] (1) Integrate the hypoxia-specific gene expression cassette into the alr gene locus on the chromosome of the SWT007 strain

[0868] 1. Construction of dadX gene knockout attenuated bacteria (SWT007)

[0869] (1) A single clone of SWT003 was selected and inoculated into 5 ml of LB liquid culture medium, and then placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0870] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0871] (3) Wash the cells three times with sterilized purified water.

[0872] (4) Preparation of PCR products of SWTO78 and SWTO79.

[0873] Primers SWTO78 and SWTO79 were mixed with plasmid pSWT002 (same as above, plasmid description), and high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0874] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0875] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0876] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO78 and SWTO79 and electroporated at a voltage of 1.8 kV.

[0877] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0878] (7) Positive clones were identified by colony PCR, and SWTO59, SWTO5, SWTO6, and SWTO87 were used to identify the insertion of the chloramphenicol resistance gene, as shown in Figure 81.

[0879] (8) The positive single clone was inoculated into 5 ml of LB medium and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0880] 2. Construction of a plasmid containing a hypoxia-specific gene expression cassette and the essential gene being the Salmonella dadX gene

[0881] (1) Use a pipette tip to pick up a single clone of the Salmonella strain SWT001 and mix it with primers SWTO39, SWTO40 and a high-fidelity PCR amplification enzyme system, and then place the mixture into a PCR amplification device.

[0882] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 60 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0883] (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 FIG2 .

[0884] (3) The recovered product was mixed with HindIII and XhoI enzyme digestion system, incubated at 37°C for 1 hour, and the digestion product was purified and recovered using a DNA gel recovery system.

[0885] (4) Take 5 μg of plasmids pOL1001, pOL1002, pOL1004, pOL1005, pOL1009, pOL1010, pOL1012, pOL1013, pOL1021, pOL1025, pOL1033, pOL1035, pOL1036, pOL1038 and pOL1063, respectively, mix them with HindIII and XhoI restriction enzyme systems, incubate at 37°C for 1 hour, recover the vector fragments and mix them with the fragments in step (3), and ligate them under the action of T4 DNA ligase.

[0886] (5) Transform into DH10B competent cells and spread on chloramphenicol and ampicillin plates to select single clones.

[0887] (6) The successfully constructed vectors were pOL2001, pOL2002, pOL2004, pOL2005, pOL2009, pOL2010, pOL2012, pOL2013, pOL2021, pOL2025, pOL2033, pOL2035, pOL2036, pOL2038, and pOL2063, as shown in Table 4.

[0888] Table 4 Combinations of forward hypoxia promoter and reverse hyperoxia promoter

[0889] The essential gene for survival in Table 4 is the dadX gene of Salmonella.

[0890] 3. Construction of defective attenuated bacteria containing a hypoxia-specific gene expression cassette and the essential gene for survival being the Salmonella dadX gene

[0891] (1) A single clone of the dadX gene-knockout attenuated bacteria SWT007 was selected and inoculated into 5 ml of LB liquid culture medium, and then placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0892] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0893] (3) Wash the cells three times with sterilized purified water.

[0894] (4) Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette. Mix primers SWTO85 and SWTO86 with a hypoxia-specific gene expression cassette plasmid (as shown in Table 4) and a high-fidelity PCR amplification enzyme system and place them in a PCR amplification device.

[0895] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 120 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0896] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0897] (5) The recovered bacteria were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV.

[0898] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine and cultured overnight in a 32°C constant temperature incubator until a single clone colony was grown.

[0899] (7) The positive single clone was inoculated into 5 ml of LB medium and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0900] (8) Using the corresponding primers, clones were identified by colony PCR. The identification results are shown in Figure 82. The correspondence between strains and identification primers is shown in Table 5.

[0901] Table 5 Correspondence between strains and identification primers

[0902] (II) Oxygen adaptability verification test of a library of Salmonella strains containing a hypoxia-specific gene expression cassette and the essential survival gene Salmonella dadX (dadX gene knockout, expression cassette integrated into the Salmonella alr gene)

[0903] (1) A single clone of the strain was selected and inoculated into 5 ml of LB liquid medium supplemented with D-alanine, and cultured in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0904] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0905] (3) Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0906] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine, mark it as "1", and repeat three times, mark it as "a", "b", and "c".

[0907] (5) After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0908] (6) Repeat this 10-fold gradient dilution until the mark “6” is reached.

[0909] (7) One plate was cultured at 37°C in an anaerobic environment, and the other plate was cultured at 37°C in an atmospheric environment (21% oxygen concentration).

[0910] The results of the oxygen adaptability verification test of the strain containing the hypoxia-specific expression cassette are shown in Figures 83-86.

[0911] The above results show that the essential gene in the hypoxia-specific gene expression cassette can be alr or dadX, both of which can have similar oxygen regulation effects.

[0912] Example 4: Study on the replacement of the source bacteria for constructing a positive hypoxia promoter

[0913] In order to supplement and expand the same positive hypoxia promoter from bacteria of different origins, the present invention designed this embodiment.

[0914] (1) Positive hypoxia promoter yhbU

[0915] In the present invention, the positive hypoxia promoter used by strains SWT1001, SWT1002, SWT1004, and SWT1005 is yhbU from Salmonella, and the positive hypoxia promoter used by strains SWT1065, SWT1066, SWT1068, and SWT1069 is yhbU from Escherichia coli.

[0916] For Gram-negative facultative anaerobes, the promoters of these bacteria all contain conserved FNR binding sites within the yhbU region. These FNR binding sites conform to the sequence "CTGCCNNNNATCAA," where N represents any of the bases A, T, C, or G. The yhbU promoter also conforms to the positive hypoxic promoter sequence "TTGATNNNNATCAA," where any base within the TTGAT and ATCAA sequences within the conserved binding site can be replaced, but the total number of replacements must not exceed three, and the replacements cannot be three consecutive bases.

[0917] The present invention verifies the yhbU promoters of Salmonella and Escherichia coli, and the results show that yhbU promoters that conform to this conserved site all have corresponding oxygen regulation functions and can be used as the positive hypoxia promoter of the hypoxia-specific gene expression cassette of the present invention.

[0918] 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), Cronobacter (SEQ ID No. 52), Klebsiella (SEQ ID No. 53), Pantoea (SEQ ID No. 54), Serratia (SEQ ID No. 55) or Simihuisella (SEQ ID No. 56).

[0919] By analyzing the sequences of the yhbU promoters from the above-mentioned different sources of bacteria, it was found that although the sequences of the promoters were quite different, their FNR binding sites were exactly the same, both being CTGCCTTAAATCAA.

[0920] Validation of oxygen adaptability of strains containing the hypoxia-specific gene expression cassette of Escherichia coli yhbU

[0921] (1) Single clones of SWT1065, SWT1066, SWT1068, and SWT1069 strains were selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0922] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0923] (3) Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0924] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine, mark it as "1", and repeat three times, mark it as "a", "b", and "c".

[0925] (5) After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0926] (6) Repeat this 10-fold gradient dilution until the mark “6” is reached.

[0927] (7) One plate was cultured at 37°C in an anaerobic environment, and the other plate was cultured at 37°C in an atmospheric environment (21% oxygen concentration).

[0928] The results of the oxygen adaptability verification test of Salmonella strains SWT1065, SWT1066, SWT1068 and SWT1069 are shown in Figure 87.

[0929] The results showed that replacing the positive hypoxia promoter from Salmonella source to Escherichia coli source can achieve similar oxygen regulation effects.

[0930] (II) Positive hypoxia promoter ynfK

[0931] The positive hypoxia promoter used by strains SWT1009, SWT1010, SWT1012, and SWT1013 is ynfK from Escherichia coli.

[0932] The positive hypoxia promoter used by strains SWT1041, SWT1042, SWT1044 and SWT1045 is ynfK from Salmonella.

[0933] For Gram-negative facultative anaerobes, the promoters of ynfK all contain conserved FNR binding sites with the sequence "TTGCGNNNNCTCAA," where N represents any of A, T, C, or G. The ynfK promoter conforms to the positive hypoxic promoter sequence "TTGATNNNNATCAA," where any base within the TTGAT and ATCAA sequences within the conserved binding site can be replaced, but the total number of replacements must not exceed three, and the replacements cannot be three consecutive bases.

[0934] The present invention verifies the ynfK promoter of Salmonella and Escherichia coli, and the results show that the ynfK that conforms to the conserved site has the corresponding oxygen regulation function and can be used as the positive hypoxia promoter of the hypoxia-specific gene expression cassette of the present invention.

[0935] 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) or Enterobacter reuteri (SEQ ID No. 59).

[0936] Validation of oxygen adaptability of strains containing a hypoxia-specific gene expression cassette from the Salmonella ynfK promoter

[0937] (1) Single clones of strains SWT1041, SWT1042, SWT1044, and SWT1045 were selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0938] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0939] (3) Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0940] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine, mark it as "1", and repeat three times, mark it as "a", "b", and "c".

[0941] (5) After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0942] (6) Repeat this 10-fold gradient dilution until the mark “6” is reached.

[0943] (7) One plate was cultured at 37°C in an anaerobic environment, and the other plate was cultured at 37°C in an atmospheric environment (21% oxygen concentration).

[0944] The results of the oxygen adaptability validation test for Salmonella strains SWT1041, SWT1042, SWT1044, and SWT1045 are shown in Figure 88. The results show that replacing the forward hypoxia promoter from E. coli with that from Salmonella can achieve similar oxygen regulation effects.

[0945] The above results show that, for example, the yhbU and ynfK promoters share highly conserved FNR binding sites across diverse facultative anaerobic bacteria. Comparison of the two promoters in Escherichia coli and Salmonella, when placed within a hypoxia-specific gene expression cassette, revealed consistent oxygen regulation. This suggests that promoters from the same gene in different facultative anaerobic bacteria, containing conserved FNR binding site fragments, can be used to construct the hypoxia-specific gene expression cassette described herein, achieving the same effect.

[0946] Example 5: Study on the replacement of the source bacteria for constructing the reverse high oxygen promoter

[0947] In order to supplement and expand the same reverse hyperoxia promoter from bacteria of different origins, the present invention designed this embodiment.

[0948] (1) Reverse high oxygen promoter ydcI

[0949] In the present invention, the reverse hyperoxia promoter used by strains SWT1005, SWT1013 and SWT1021 is the ydcI promoter of Salmonella.

[0950] The reverse hyperoxia promoter used by strains SWT1008, SWT1016, and SWT1024 was the ydcI promoter of Escherichia coli.

[0951] The ydcI promoters of Gram-negative facultative anaerobes all contain conserved FNR binding sites with the sequence "CTGCCNNNNATCAA," where N represents any of A, T, C, or G. The ydcI promoter conforms to the reverse hyperoxic promoter pattern of "TTGATNNNNATCAA," where any base within the conserved binding site, TTGAT and ATCAA, can be replaced, but the total number of replacements cannot exceed three, and three consecutive bases cannot be replaced. Furthermore, the ArcA binding site of the reverse hyperoxic promoter conforms to the pattern of GTTAATTA, where any base can be replaced, but the total number of replacements cannot exceed two.

[0952] The present invention verifies the ydcI promoters of Salmonella and Escherichia coli, and the results show that the ydcI promoters that conform to the conserved site all have corresponding oxygen regulation functions and can be used as the reverse high oxygen promoter of the hypoxia-specific gene expression cassette of the present invention.

[0953] 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).

[0954] Validation of oxygen adaptability of a hypoxia-specific gene expression cassette containing the Escherichia coli ydcI promoter

[0955] (1) Single clones of strains SWT1008, SWT1016, and SWT1024 were selected and inoculated into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0956] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0957] (3) Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0958] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine, mark it as "1", and repeat three times, mark it as "a", "b", and "c".

[0959] (5) After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0960] (6) Repeat this 10-fold gradient dilution until the mark “6” is reached.

[0961] (7) One plate was cultured at 37°C in an anaerobic environment, and the other plate was cultured at 37°C in an atmospheric environment (21% oxygen concentration).

[0962] The results of the oxygen adaptability verification test of Salmonella strains SWT1008, SWT1016 and SWT1024 are shown in Figure 89.

[0963] The results showed that replacing the forward high oxygen promoter from Salmonella source to Escherichia coli source can achieve similar oxygen regulation effects.

[0964] (ii) The reverse hyperoxia promoter mqo containing two ArcA binding sites

[0965] In the present invention, the reverse hyperoxia promoter used by strains SWT1004, SWT1012 and SWT1036 is the argT promoter of Salmonella (SEQ ID No. 12).

[0966] The reverse hyperoxia promoter used by strains SWT1073, SWT1074, and SWT1075 was the mqo promoter of Escherichia coli (SEQ ID No. 62).

[0967] Both the argT promoter (SEQ ID No. 12) and the mqo promoter (SEQ ID No. 62) contain conserved double ArcA binding sites and conform to the ArcA binding site of a reverse hyperoxia promoter, conforming to the GTTAATTA pattern, wherein any base can be replaced, but the total number of replacements does not exceed 2. The present invention validated the argT promoter (SEQ ID No. 12) and the mqo promoter (SEQ ID No. 62), and the results demonstrated that promoters conforming to the conserved double ArcA binding site have corresponding oxygen regulation functions and can serve as reverse hyperoxia promoters for the hypoxia-specific gene expression cassette of the present invention.

[0968] Therefore, the inverted hyperoxia promoter used in the hypoxia-specific gene expression cassette designed in the present invention can be derived from the argT promoter (SEQ ID No. 12) and the mqo promoter (SEQ ID No. 62) containing double ArcA binding sites.

[0969] Validation of oxygen adaptability of hypoxia-specific gene expression cassettes containing the mqo promoter

[0970] (1) Single clones of strains SWT1073, SWT1074, and SWT1075 were selected and inoculated into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0971] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0972] (3) Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0973] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine, mark it as "1", and repeat three times, mark it as "a", "b", and "c".

[0974] (5) After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0975] (6) Repeat this 10-fold gradient dilution until the mark “6” is reached.

[0976] (7) One plate was cultured at 37°C in an anaerobic environment, and the other plate was cultured at 37°C in an atmospheric environment (21% oxygen concentration).

[0977] The results of the oxygen adaptability verification test of Salmonella strains SWT1073, SWT1074 and SWT1075 are shown in Figure 90.

[0978] The results showed that replacing the forward high oxygen promoter from Salmonella source to Escherichia coli source can achieve similar oxygen regulation effects.

[0979] The specific sequences of the forward hypoxia promoter, the survival essential gene and the reverse hyperoxia promoter of the present invention are shown in Table 6.

[0980] Table 6 Specific sequences of the forward hypoxia promoter, essential survival genes, and reverse hyperoxia promoter of the present invention

[0981] The specific sequences of all primers used in the present invention are shown in Table 7.

[0982] Table 7 Specific sequences of all primers used in the present invention

[0983] The tool plasmids of the present invention are shown in Table 8.

[0984] Table 8 Tool plasmids used in the present invention

[0985] The present application has been described above in conjunction with preferred embodiments, but these embodiments are merely exemplary and serve only as an illustrative example. On this basis, various replacements and improvements can be made to the present application, all of which fall within the scope of protection of the present application.

Claims

1. A hypoxia-specific gene expression cassette, comprising: a) a positive hypoxia promoter, which is a promoter comprising an FNR binding site, and the positive hypoxia promoter can be induced to express under hypoxia; b) genes essential for survival; and c) a reverse hyperoxic promoter, which is a promoter comprising FNR and ArcA binding sites, and the reverse hyperoxic promoter can function under the oxygen content conditions of normal organs; The survival essential gene is a gene encoding alanine racemase.

2. A hypoxia-specific gene expression cassette, comprising: a) a positive hypoxia promoter, which is a promoter comprising an FNR binding site, and the positive hypoxia promoter can be induced to express under hypoxia; b) genes essential for survival; and c) an inverted hyperoxic promoter, which is a promoter comprising two ArcA binding sites, and the inverted hyperoxic promoter is able to function under the oxygen content conditions of normal organs; The survival essential gene is a gene encoding alanine racemase.

3. The hypoxia-specific gene expression cassette according to claim 1 or 2, wherein: The FNR binding site of the forward hypoxia promoter or the reverse hyperoxia promoter conforms to the pattern of TTGATNNNNATCAA, where N is any base among A, T, C, and G. Any base in the TTGAT and ATCAA sequences in the conserved binding site can be replaced, but the total number of replacements does not exceed 3, and three consecutive adjacent bases cannot be replaced; The ArcA binding site of the inverse hyperoxia promoter conforms to the pattern of GTTAATTA, in which any base can be replaced, but the total number of replacements does not exceed 2.

4. The hypoxia-specific gene expression cassette according to any one of the preceding claims, wherein The positive hypoxia promoter is selected from the promoter region sequence of yhbU, ynfK, tdcA, yecH or focA; Genes essential for survival are selected from alr or dadX; The reverse hyperoxia promoter is selected from the promoter region of cyoA, ydcI, phoH, argT, mqo or lldP domain sequence. The hypoxia-specific gene expression cassette according to claim 4 , wherein the expression cassette is composed of a forward hypoxia promoter yhbU, a survival essential gene alr, and a reverse hyperoxia promoter cyoA. 6 . The hypoxia-specific gene expression cassette according to claim 4 , wherein the expression cassette is composed of a forward hypoxia promoter yhbU, a survival essential gene alr, and a reverse hyperoxia promoter ydcI.

7. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yhbU, a survival essential gene alr and a reverse hyperoxia promoter phoH.

8. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette is composed of a forward hypoxia promoter yhbU, a survival essential gene alr, and a reverse hyperoxia promoter argT.

9. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette is composed of a forward hypoxia promoter yhbU, a survival essential gene alr, and a reverse hyperoxia promoter mqo. 10 . The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter ynfK, a survival essential gene alr, and a negative hyperoxia promoter cyoA. The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter ynfK, a survival essential gene alr, and a negative hyperoxia promoter phoH. 12 . The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter ynfK, a survival essential gene alr, and a negative hyperoxia promoter argT. 13 . The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter ynfK, a survival essential gene alr, and a negative hyperoxia promoter ydcI. The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter ynfK, a survival essential gene alr, and a negative hyperoxia promoter mqo. The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter tdcA, a survival essential gene alr, and a negative hyperoxia promoter ydcI. The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter tdcA, a survival essential gene alr, and a negative hyperoxia promoter cyoA. 17 . The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a forward hypoxia promoter yecH, a survival essential gene alr, and a reverse hyperoxia promoter cyoA.

18. The hypoxia-specific gene expression cassette according to claim 4, wherein the expression cassette is composed of a forward hypoxia promoter yecH, a survival essential gene alr, and a reverse hyperoxia promoter lldP. The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter yecH, a survival essential gene alr and a negative hyperoxia promoter argT.

20. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a positive hypoxia promoter yecH, a survival essential gene alr, and a negative hyperoxia promoter phoH.

21. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yecH, a survival essential gene alr and a reverse hyperoxia promoter mqo.

22. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter focA, a survival essential gene alr, and a reverse hyperoxia promoter lldP.

23. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yhbU, a survival essential gene dadX and a reverse hyperoxia promoter cyoA. The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperoxia promoter ydcI.

25. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperoxia promoter phoH.

26. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yhbU, a survival essential gene dadX, and a reverse hyperoxia promoter argT.

27. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yhbU, a survival essential gene dadX and a reverse hyperoxia promoter mqo.

28. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a positive hypoxia promoter ynfK, a survival essential gene dadX, and a negative hyperoxia promoter cyoA.

29. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a positive hypoxia promoter ynfK, a survival essential gene dadX, and a negative hyperoxia promoter phoH.

30. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a positive hypoxia promoter ynfK, a survival essential gene dadX, and a negative hyperoxia promoter argT.

31. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a positive hypoxia promoter ynfK, a survival essential gene dadX, and a negative hyperoxia promoter ydcI.

32. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperoxia promoter mqo.

33. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a positive hypoxia promoter tdcA, a survival essential gene dadX and a negative hyperoxia promoter ydcI. The hypoxia-specific gene expression cassette according to claim 4 , which is composed of a positive hypoxia promoter tdcA, a survival essential gene dadX, and a negative hyperoxia promoter cyoA.

35. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yecH, a survival essential gene dadX and a reverse hyperoxia promoter cyoA.

36. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yecH, a survival essential gene dadX and a reverse hyperoxia promoter lldP.

37. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yecH, a survival essential gene dadX and a reverse hyperoxia promoter argT.

38. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a positive hypoxia promoter yecH, a survival essential gene dadX and a negative hyperoxia promoter phoH.

39. The hypoxia-specific gene expression cassette according to claim 4, which is composed of a forward hypoxia promoter yecH, a survival essential gene dadX and a reverse hyperoxia promoter mqo.

40. The hypoxia-specific gene expression cassette of claim 4, wherein the expression cassette is composed of a forward hypoxia promoter focA, a survival essential gene dadX, and a reverse hyperoxia promoter lldP.

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

42. The hypoxia-specific gene expression cassette of claim 41, wherein the gram-negative bacteria is selected from the group consisting of Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simihuisella, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae Bacteria, Vibrio parahaemolyticus, Vibrio cholerae, Yersinia pestis, Coccus catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia and Helicobacter pylori.

43. A hypoxia-specific gene expression cassette according to any of the above claims, wherein the positive hypoxia promoter is yhbU, which is selected from Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia and Simhuisella.

44. The hypoxia-specific gene expression cassette according to any one of the preceding claims, wherein the positive hypoxia promoter is ynfK, which is selected from the group consisting of Salmonella, Escherichia coli, Serratia, Shigella and Enterobacter reuteri.

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

46. ​​The hypoxia-specific gene expression cassette according to any one of the preceding claims, which is regulated by oxygen concentration.

47. The hypoxia-specific gene expression cassette of claim 46, wherein the forward hypoxia promoter functions when the oxygen content is below 1%, but fails to function when the oxygen content is above 1%; and / or the reverse hyperoxia promoter functions when the oxygen content is above 1%, but fails to function when the oxygen content is below 1%.

48. The hypoxia-specific gene expression cassette of claim 47, wherein the forward hypoxia promoter functions when the oxygen content is below 0.8%, but fails to function when the oxygen content is above 0.8%; and / or the reverse hyperoxia promoter functions when the oxygen content is above 0.8%, but fails to function when the oxygen content is below 0.8%.

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

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

51. The Gram-negative bacteria according to claim 50, which is Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simihuierella, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida, Legionella, Klebsiella pneumoniae, Parahaemolyticus Vibrio, Vibrio cholerae, Yersinia pestis, Coccus catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolytica, Salmonella enterica, Salmonella bongoli, Salmonella paratyphi, Salmonella typhi, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, or Helicobacter pylori.

52. The Gram-negative bacterium according to claim 51, which is Salmonella.

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

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

55. The pharmaceutical composition of claim 54, wherein the pharmaceutically acceptable carrier is selected from the group consisting of disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants and lubricants.

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

57. Use of the hypoxia-specific gene expression cassette of any one of claims 1 to 49, the Gram-negative bacteria of any one of claims 50 to 52, or the pharmaceutical composition of any one of claims 53 to 56 in the preparation of tumor drugs.

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

59. According to the pharmaceutical composition of claim 56 or the use of claim 58, the solid tumor is selected from the group consisting of tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestine (colon cancer, rectal cancer), pelvic cavity (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer, and bladder.

60. According to the pharmaceutical composition of claim 56 or the use of claim 58, the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

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

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

63. The method of claim 62, wherein the solid tumor is selected from the group consisting of tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestine (colon cancer, rectal cancer), pelvic cavity (cervical cancer, ovarian malignancies, endometrial cancer, ovarian cancer), nervous system, head and neck cancer, and bladder.

64. The method of claim 63, wherein the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma, and bladder cancer.

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