Hypoxia specific gene expression cassette, bacteria containing hypoxia specific gene expression cassette and application of hypoxia specific gene expression cassette

Abstract

The invention belongs to the field of gene engineering and biological treatment, and provides a hypoxia specific gene expression cassette, gram-negative bacteria containing the expression cassette and application of the gram-negative bacteria in preparation of tumor drugs. According to the invention, the forward hypoxia promoter and the reverse hyperxia promoter are reasonably designed, and appropriate survival essential genes are matched, so that the hypoxia specific gene expression cassette which is more efficient and safer is obtained. Gram-negative bacteria modified by the hypoxia specific gene expression cassette designed by the invention are low in overall mutation rate, and in a mouse in-vivo tumor model, more bacteria are distributed in tumors and the clearing speed in normal organs is higher.

Description

Technical Field

[0001] The invention relates to a hypoxia-specific gene expression box, bacteria containing the same and application thereof, and belongs to the field of genetic engineering and biological treatment. Background Art

[0002] Among all malignant tumors, solid tumors account for about 90%, such as sarcoma, melanoma, breast cancer, lung cancer, colon cancer, prostate cancer, etc.

[0003] The tumor microenvironment of solid tumors has common characteristics, including abnormal tumor vasculature, excessive connective tissue, immunosuppression, acidic environment and hypoxic areas. In addition, the abnormal microenvironment of solid tumors is a natural barrier that is difficult for traditional therapeutic drugs to penetrate. Chemotherapy drugs and antibody drugs are difficult to diffuse in the solid tumor microenvironment. In addition, the lack of oxygen free radicals makes the tumor resistant to chemotherapy and radiotherapy.

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

[0005] As early as 1868, German doctor W. Busch first reported that in some tumor patients, when they were infected with bacteria (Streptococcus pneumoniae), the growth of tumors in their bodies was inhibited or even completely eliminated. In the following thirty years, American doctor Coley and another German doctor Fehleisen reported that bacterial infection can inhibit tumors. These early studies have been controversial because the results are difficult to replicate and the virulence of bacteria is difficult to control. However, rigorous animal experiments later proved that bacterial infection can indeed reduce the size of tumors, and the host's immune system will also be activated during the treatment process. In 1975, Carswell first reported that lipopolysaccharide of Gram-negative bacteria can stimulate the immune system to release tumor necrosis factor TNF-α and can cause the death of tumor cells. In addition, some bacterial vaccines have also been shown to stimulate the immune system to treat tumors. Among them, BCG (BCG) is the earliest biological agent used in clinical tumor treatment. BCG is a live attenuated bacterial suspension made by continuous inoculation of highly pathogenic bovine tuberculosis bacteria for 230 generations, and its purpose is to prevent tuberculosis. A large number of experiments and clinical practices have confirmed that BCG is one of the most effective means of treating bladder cancer.

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

[0007] Studies have shown that after attenuation of Salmonella typhimurium by different methods, its colonization ability in tumor tissue can reach 1000-10000 times that of normal tissue. The methods of strain attenuation include:

[0008] 1. AroA gene defect, strain SL7207. The aroA mutant strain cannot synthesize para-aminobenzoic acid (PABA) and aromatic amino acids by itself, and the content of PABA in the body environment is low, thereby reducing its toxicity in the body.

[0009] 2. Defects in the purI and msbB genes, strain VNP20009. The mutation of purI prevents the synthesis of adenosine monophosphate and reduces toxicity in vivo; the mutation of msbB can prevent the synthesis of lipid A in cell wall lipopolysaccharide, thereby avoiding bacterial infection sepsis caused by excessive secretion of TNF-α. In previous animal experiments, this strain can inhibit tumor growth, and its safety as a live bacterial intravenous administration has been proven in Phase I clinical trials.

[0010] 3. Leucine and arginine deficiency, strains A1 and A1-R. Zhao et al. induced mutagenesis and screened A1 with chemical reagents, which was able to inhibit the growth of tumor cells in both in vivo and in vitro experiments. In order to enhance its tumor targeting ability, Zhao et al. re-isolated A1 from the inside of the tumor to obtain the A1-R strain with 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 (mitomycin C) and colicin E3 (colicin E3) were loaded into VNP20009 for therapeutic experiments in various tumor models.

[0011] Although the above-mentioned nutritional deficiency method can significantly reduce the distribution of bacteria in normal organs, such as liver, spleen, etc., it is difficult to see a significant decrease in the distribution of bacteria in normal organs over time.

[0012] Salmonella YB1 is a tumor-targeting strain developed based on the above research foundation, combined with chromosome homologous recombination technology and the concept of synthetic biology. It has a preference for tumor hypoxic areas and tumor accumulation ability.

[0013] 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 achieving survival and growth in the tumor under hypoxic conditions and reducing accumulation in normal organs. Compared with ordinary oncolytic bacteria, this method increases safety.

[0014] YB1 is the first generation of attenuated Salmonella based on oxygen-regulated promoters designed from the analysis of oxygen in tumors. Therefore, in terms of its mechanism, it is significantly superior to ordinary nutritionally deficient strains. However, due to the easy mutation of bacteria, 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 likely to evolve in the absence of diaminopimelic acid (DAP), thus 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 -6 Each 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 Every year, the division rate of Salmonella is 20-30 minutes per generation, which is equivalent to the mutation frequency of YB1 being 10,000 times higher than the natural mutation frequency of Salmonella. Therefore, the substantial increase in the mutation frequency of YB1 is an abnormal phenomenon. This abnormal high-frequency mutation reduces the advantage of hypoxia regulation, which may affect its safety and may also bring new risks to tumor treatment. The reason may be due to the unreasonable regulation of the gene elements pepT promoter, asd gene, and sodA promoter. Therefore, the new regulation mode is very important.

[0015] According to a 2012 study on YB1, 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. It takes up to 26 days to be completely eliminated from most normal organs of mice, but a small amount of residue still exists in the liver. The reason may be related to the high evolution rate caused by the design defects of YB1 itself.

[0016] In summary, there is an urgent need in this field for 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. Summary of the invention

[0017] In the present invention, unless otherwise specified, the scientific and technical terms used in the present invention have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures of protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in the present invention are the terms and conventional procedures widely used in the corresponding fields.

[0018] In view of the above-mentioned defects in the prior art, the present invention provides a hypoxia-specific gene expression cassette, which comprises:

[0019] 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;

[0020] b) genes essential for survival; and

[0021] 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;

[0022] The survival essential gene is a gene encoding alanine racemase.

[0023] Another aspect of the present invention provides a hypoxia-specific gene expression cassette comprising:

[0024] 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;

[0025] b) genes essential for survival; and

[0026] 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;

[0027] The survival essential gene is a gene encoding alanine racemase.

[0028] "Polypeptide", "peptide", and "protein" are used interchangeably in the present invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acids, as well as to polymers of natural amino acids. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0029] In the present invention, "polynucleotide" refers to a macromolecule formed by connecting multiple nucleotides through phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequence of the polynucleotide of the present invention can be codon-optimized for different host cells (such as Escherichia coli) to improve the expression of the polypeptide. Methods for codon optimization are known in the art.

[0030] In order for bacteria to survive only in low oxygen concentration conditions, three requirements need to be met:

[0031] 1) Control the strength of the upstream promoter and the background leakage expression.

[0032] If the oxygen sensor partially leaks expression without binding to the upstream promoter, it will make it impossible to regulate oxygen, resulting in the bacteria surviving at any oxygen concentration; or if the strength of the upstream promoter is too weak, it will not be able to start the expression of the downstream gene, resulting in the bacteria not surviving at any oxygen concentration;

[0033] 2) Select appropriate genes essential for survival.

[0034] By selecting genes essential for survival, it can be ensured that the bacteria die when the genes essential for survival are not expressed, and the survival of the bacteria can be quickly ensured when the expression is induced by hypoxia;

[0035] 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.

[0036] Therefore, in the present invention, "hypoxia-specific gene expression cassette" refers to a piece of DNA that can initiate expression of essential genes 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.

[0037] In the present invention, "essential gene" refers to a gene that plays a decisive role in the growth and / or survival of bacteria. Once the bacteria lack this gene or its functional expression product, they cannot survive, divide and / or grow normally. A typical example of lacking an essential gene or its functional expression product is a nutrient-deficient strain, which cannot survive, divide and / or grow normally under in vitro culture conditions or in vivo environments in the absence of specific exogenous supplements. Essential genes usually exist in a single copy on bacterial chromosomes.

[0038] From this, we can see that the requirements for genes essential for survival are as follows:

[0039] (1) It is an essential gene for bacterial reproduction. If it is missing, it will lead to rapid death of bacteria;

[0040] (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 facilitate the cultivation and preparation of bacteria in a normal culture environment by adding the corresponding expression product of this gene;

[0041] (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.

[0042] In the present invention, the survival essential gene is a gene encoding alanine racemase.

[0043] For Gram-negative bacteria such as Escherichia coli and Salmonella, the cell wall is an indispensable component. The core component of the cell wall is peptidoglycan. When synthesizing peptidoglycan, bacteria need D-alanine as an important component. If D-alanine is missing, the bacteria will not be able to synthesize the cell wall and will then lyse.

[0044] All amino acids that 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.

[0045] 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.

[0046] In our previous study, YB1 Salmonella used asd gene as essential gene for regulation. Compared with YB1 Salmonella, the present invention selected alr gene and dadX gene as essential genes for survival. alr gene and dadX gene are functionally homologous genes. Therefore, in the present invention, the gene of alanine racemase can be the alr gene (SEQ ID No. 17) and the protein sequence of alr of Salmonella (SEQ ID No. 18) or the dadX gene (SEQ ID No. 19) and the protein sequence of dadX of Salmonella (SEQ ID No. 20); or the alr gene (SEQ ID No. 21) and the protein sequence of alr of Escherichia coli (SEQ ID No. 22) or the dadX gene (SEQ ID No. 23) and the protein sequence of dadX of Escherichia coli (SEQ ID No. 24); or the alr gene (SEQ ID No. 25) and the protein sequence of alr of Shigella (SEQ ID No. 26) or the dadX gene (SEQ ID No. 27) and the protein sequence of dadX of Shigella (SEQ ID No. 28); or the alr gene (SEQ ID No. 28) of Klebsiella No.29) and the protein sequence of Klebsiella alr (SEQ ID No.30) or Klebsiella dadX gene (SEQ ID No.31) and the protein sequence of Klebsiella dadX (SEQ ID No.32); or the alr gene (SEQ ID No.33) of Yersinia and the protein sequence of Yersinia alr (SEQ ID No.34) or the dadX gene (SEQ ID No.35) of Yersinia dadX (SEQ ID No.36); or the alr gene (SEQ ID No.37) of Haemophilus and the protein sequence (SEQ ID No.38) of Haemophilus alr; or the alr gene (SEQ ID No.39) of Pseudomonas and the protein sequence (SEQ ID No.40) of Pseudomonas alr or the dadX gene (SEQ ID No.41) of Pseudomonas dadX (SEQ ID No.42) of Pseudomonas.

[0047] In the present invention, alr gene knockout bacteria were first constructed, and then another gene dadX was modified, so that the bacteria became alr gene and dadX gene defective bacteria after editing, and the positive hypoxia promoter pepT and the reverse hyperoxia promoter sodA of YB1 were used to regulate the additional alr gene (SWT006). According to the experimental results (SWT006 verification experiment), the alr gene and dadX gene were selected as the means of regulating essential genes, and their controllability was higher than the essential gene asd previously selected by YB1 Salmonella.

[0048] The positive hypoxia promoter of the present invention is a hypoxic promoter regulated by FNR; the reverse hyperoxic promoter of the present invention is an antisense promoter negatively regulated by FNR and / or ArcA. The fumarate and nitrate reduction gene fnr is an important gene regulating the aerobic and anaerobic growth of Salmonella. This complex regulatory system has been widely studied in Escherichia coli and Salmonella. The DNA binding protein FNR encoded by the fnr gene senses changes in oxygen and controls the expression of different genes, achieving switching at the overall metabolic level. Therefore, DNA binding sequences such as FNR and ArcA become an important way to control the expression of downstream genes.

[0049] FNR has an oxygen-sensitive [4Fe-4S] 2+ The domain can directly sense oxygen and regulate site-specific DNA binding. In contrast, ArcA senses signals from the aerobic respiratory chain. Therefore, there are two different mechanisms for sensing changes in oxygen concentration in Gram-negative facultative anaerobes such as Salmonella and Escherichia coli. When FNR regulates gene expression in hypoxia, it is divided into two situations: activation and inhibition.

[0050] To this end, the present invention screened out the forward hypoxia promoters yhbU, ynfK, yecH, cydA, focA and tdcA that FNR activates the expression of downstream genes under hypoxia, and the reverse hyperoxia promoters ydcI, cyoA, phoH, argT, mqo and lldP ​​that FNR and / or ArcA inhibit the expression of downstream genes under hypoxia. In addition, promoters from different sources were screened as the forward hypoxia promoter and the reverse hyperoxia promoter used in the present invention.

[0051] Among them, the positive hypoxia promoter used in the present invention is as follows:

[0052] (1) Salmonella yhbU promoter (yhbU-S) (SEQ ID No. 1), wherein the FNR binding site is "CTGCCTTAAATCAA";

[0053] (2) Escherichia coli ynfK promoter (ynfK-E) (SEQ ID No. 2), wherein the FNR binding site is "TTGCGCTATCTCAA";

[0054] (3) Salmonella tdcA promoter (tdcA-S) (SEQ ID No. 3), wherein the FNR binding site is "TTGATTGAAATCAG";

[0055] (4) Escherichia coli tdcA promoter (tdcA-E) (SEQ ID No. 4), wherein the FNR binding site is "TTGACAAAAATCAG";

[0056] (5) Escherichia coli yecH promoter (yecH-E) (SEQ ID No. 5), wherein the FNR binding site is "TTCATAAGCGGCAA";

[0057] (6) Salmonella ynfK promoter (ynfK-S) (SEQ ID No. 6), wherein the FNR binding site is "TTGCGCTAACTCAA";

[0058] (7) Escherichia coli cydA promoter (cydA-E) (SEQ ID No. 7), wherein the FNR binding site is "TTGATATTTATCAA";

[0059] (8) Escherichia coli focA promoter (focA-E) (SEQ ID No. 8), wherein the FNR binding site is "ATGATCTATATCAA".

[0060] Among them, the reverse high oxygen promoter used in the present invention is as follows:

[0061] (1) Salmonella cyoA promoter (cyoA-S) (SEQ ID No. 9), wherein the binding site of FNR is "TTTATTGATAATAA" and the binding site of ArcA is "GTTAAGTA";

[0062] (2) Salmonella phoH promoter (phoH-S) (SEQ ID No. 10), wherein the binding site of FNR is "TTGAGCGCCGTTAA" and the binding site of ArcA is "GTTAATTA";

[0063] (3) Salmonella lldP ​​promoter (lldP-S) (SEQ ID No. 11), wherein the binding site of FNR is "TCCATACACAACAA" and the binding site of ArcA is "GTTAACTA";

[0064] (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";

[0065] (5) Salmonella ydcI promoter (ydcI-S) (SEQ ID No. 13), wherein the binding site of FNR is "GTTATCAAAAACAA" and the binding site of ArcA is "GTTAATAA";

[0066] (6) Escherichia coli phoH promoter (phoH-E) (SEQ ID No. 14), wherein the binding site of FNR is "TTCATCACTGTCAT" and the binding site of ArcA is "GTTAAATA";

[0067] (7) Escherichia coli lldP ​​promoter (lldP-E) (SEQ ID No. 15), wherein the binding site of FNR is "TTCATTGTCATTAT" and the binding site of ArcA is "TTTAGTTA";

[0068] (8) Escherichia coli ydcI promoter (ydcI-E) (SEQ ID No. 16), wherein the binding site of FNR is "GTTATCATAATCAA" and the binding site of ArcA is "GTTAATAA";

[0069] (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".

[0070] Through the 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.

[0071] 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, N is any base of A, T, C, G, and any base in the TTGAT and ATCAA sequences in the conserved binding site can be replaced, but the total number of replacements does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; the ArcA binding site of the 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.

[0072] 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.

[0073] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene alr and a negative hyperoxia promoter cyoA.

[0074] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene alr and a negative hyperoxia promoter ydcI.

[0075] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene alr and a negative hyperoxia promoter phoH.

[0076] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene alr and a negative hyperoxia promoter argT.

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

[0078] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter ynfK, a survival essential gene alr and a negative hyperoxia promoter cyoA.

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

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

[0081] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter ynfK, a survival essential gene alr and a negative hyperoxia promoter ydcI.

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

[0083] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter tdcA, a survival essential gene alr and a negative hyperoxia promoter ydcI.

[0084] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter tdcA, a survival essential gene alr and a negative hyperoxia promoter cyoA.

[0085] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yecH, a survival essential gene alr and a negative hyperoxia promoter cyoA.

[0086] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yecH, a survival essential gene alr and a negative hyperoxia promoter lldP.

[0087] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yecH, a survival essential gene alr and a negative hyperoxia promoter argT.

[0088] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yecH, a survival essential gene alr and a negative hyperoxia promoter phoH.

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

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

[0091] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene dadX and a negative hyperoxia promoter cyoA.

[0092] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene dadX and a negative hyperoxia promoter ydcI.

[0093] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene dadX and a negative hyperoxia promoter phoH.

[0094] 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.

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

[0096] In a further preferred embodiment of the present invention, the expression cassette consists of the positive hypoxia promoter ynfK, the survival essential gene dadX and the negative hyperoxia promoter cyoA.

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

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

[0099] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter ynfK, a survival essential gene dadX and a negative hyperoxia promoter ydcI.

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

[0101] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter tdcA, a survival essential gene dadX and a negative hyperoxia promoter ydcI.

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

[0103] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yecH, a survival essential gene dadX and a negative hyperoxia promoter cyoA.

[0104] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yecH, a survival essential gene dadX and a negative hyperoxia promoter lldP.

[0105] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yecH, a survival essential gene dadX and a negative hyperoxia promoter argT.

[0106] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yecH, a survival essential gene dadX and a negative hyperoxia promoter phoH.

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

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

[0109] 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 binding site for CRP, and half is a binding site for FNR. 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 conservative binding site GTGANNNNNNTCAC, and the second half CGCAA conforms to the FNR conservative region ATCAA. The A at the tenth position is replaced by C, and the T at the eleventh position is replaced by G, which does not conform to the rules for the positive hypoxia promoter in the present invention.

[0110] The FNR binding region TTGATAATCATTTT of the reverse hyperoxia promoter sodA disclosed in patent CN104471057B only contains the first half region TTGAT of FNR, while the second half region contains three consecutive substitutions of ATCAA to ATTTT, and therefore does not contain a complete FNR binding site. The ArcA binding site contained therein is TTTAATTA, and compared with the conserved core binding site 5'-GTTAATTA-3' of ArcA, the first G is replaced by T. Therefore, the promoter of sodA contains only a single ArcA binding site, and its FNR binding site is incomplete, which does not meet the rules for the reverse hyperoxia promoter in the present invention.

[0111] 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.

[0112] 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.

[0113] In the present invention, term " gram-negative bacteria " refers to after carrying out the known partial procedure of gram staining, the bacteria of the initial basic dye staining agent (such as, crystal violet) as is not retained.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 combined with basic dye (such as, crystal violet) and is trapped in cell.Then, cell is washed with acetone or ethanol, and then counterstained (such as, safranin) with the second dye of different colors.Gram-positive organism retains initial purple dyeing, and gram-negative organism is decolorized 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.

[0114] 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, Simhuisella, 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, 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.

[0115] In the present invention, the term "live bacteria" refers to a strain that has vitality, active nutritional metabolic characteristics and can perform its own biological functions. Live bacteria can include bacterial biomass produced during the metabolism of the strain.

[0116] In a further preferred embodiment of the present invention, the positive hypoxic promoter is yhbU, which is selected from the group consisting of Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia and Simhuisella.

[0117] 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.

[0118] In a preferred embodiment of the present invention, the alanine racemase gene alr and / or dadX is selected from the group consisting of Salmonella, Escherichia coli, Shigella, Klebsiella, Yersinia, Haemophilus and Pseudomonas.

[0119] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is regulated by oxygen concentration.

[0120] In order for gram-negative bacteria such as Salmonella to survive in the hypoxic zone, 1% oxygen concentration is a very important key point, which represents pathological hypoxia. Because oxygen concentrations below 1% are a clear sign of a tumor hypoxic zone, and there are no areas with oxygen concentrations below 1% in normal organ tissues. For example, the oxygen concentration in the hypoxic zone of pancreatic cancer, cervical cancer, prostate cancer and many other tumors is less than 0.7%.

[0121] The hypoxia-specific gene expression cassette designed by the present invention enables Salmonella and other Gram-negative bacteria to recognize hypoxic areas of tumors and proliferate in hypoxic areas of tumors by precisely regulating oxygen. The goal achieved by the present invention is that when the oxygen concentration is lower than 0.8%, the modified Salmonella and other Gram-negative bacteria can survive and proliferate, and achieve suicide lysis in a normal oxygen concentration environment.

[0122] Thus, in a more preferred embodiment of the present invention, the positive hypoxia promoter functions when the oxygen content is lower than 1%, but fails to function when the oxygen content is higher than 1%; and / or

[0123] The inverse hyperoxic promoter is functional when the oxygen content is above 1%, but ineffective when the oxygen content is below 1%.

[0124] 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

[0125] The inverse hyperoxic promoter is functional when the oxygen content is above 0.8%, but ineffective when the oxygen content is below 0.8%.

[0126] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is integrated into the chromosome of the host bacteria.

[0127] 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.

[0128] In a preferred embodiment of the present invention, the Gram-negative bacteria are Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simhuisella, 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.

[0129] In a more preferred embodiment of the present invention, the Gram-negative bacteria is Salmonella typhimurium.

[0130] 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.

[0131] In a preferred embodiment of the present invention, the tumor is a solid tumor.

[0132] The term "solid tumor" used in the present invention refers to an abnormal mass of tissue, which does not usually include cysts or liquid areas. Solid tumors may be benign (non-cancer), or malignant (cancer). Different types of malignant solid tumors are named after the cell types that form them. Examples of malignant solid tumors are sarcomas, carcinomas and lymphomas. Leukemia (blood cancer) does not usually form malignant solid tumors. Malignant solid tumors include but are not limited to abnormal cell masses that may be derived from different tissue types, such as liver, colon, colorectal, skin, breast, pancreas, cervix, uterine body, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testis, thyroid or lung, so malignant solid tumors include malignant solid liver tumors, colon tumors, colorectal tumors, skin tumors, breast tumors, pancreatic tumors, cervical tumors, uterine body tumors, bladder tumors, gallbladder tumors, kidney tumors, laryngeal tumors, lip tumors, oral tumors, esophageal tumors, ovarian tumors, prostate tumors, stomach tumors, testicular tumors, thyroid tumors or lung tumors, etc.

[0133] Thus, in a more preferred embodiment of the present invention, 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.

[0134] 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.

[0135] In the present invention, for the purpose of treatment, the term "subject" is preferably a subject in need of treatment of 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.

[0136] 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 with a malignant tumor, reducing the dose 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 with a malignant tumor.

[0137] 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 use, 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 presented during the development of the disease. For therapeutic use, 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 an animal model system that predicts the efficacy of the drug on human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by tests 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.

[0138] As used in the present invention, "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 range that is incompatible with the active compound, may be in the pharmaceutical composition of the present invention.

[0139] 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 live bacteria.

[0140] In a preferred embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0141] 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.

[0142] 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 the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, body surface administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0143] 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.

[0144] 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 form; 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.

[0145] Bacteria, medicine or pharmaceutical composition of the present invention can be manufactured by methods well known in the art, such as microbial growth in a fermentation tank, followed by centrifugal concentration and washing, filtration or dialysis, conventional granulation, mixing, dissolving, encapsulation, lyophilization or emulsification processes and other methods. Bacteria, medicine or pharmaceutical composition of the present invention can be produced in various forms, including particles, precipitation 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 these combinations.

[0146] The bacterium, medicine or pharmaceutical composition of the present invention can be used alone or in the presence of a carrier in combination with other compounds or compositions. In a preferred embodiment of the present invention, the bacterium, medicine or pharmaceutical composition can be used in combination with other malignant tumor therapies (including but not limited to radiotherapy, chemotherapy and surgery). The bacterium, medicine or pharmaceutical composition can be used as an adjuvant in therapy in such a case.

[0147] The present invention achieves the following beneficial effects by adopting the above technical solution:

[0148] 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.

[0149] 2. The present invention selects alr or dadX as the essential genes for survival. The controllability of alr and dadX as the essential genes for survival is higher than that of asd, the essential gene selected by YB1 Salmonella.

[0150] 3. The hypoxia-specific gene expression cassette designed by the present invention enables the modified bacteria to recognize the hypoxic area of ​​the tumor and proliferate in the hypoxic area of ​​the tumor by precisely regulating oxygen. When the oxygen concentration is lower than 0.8%, the modified bacteria transformed by the present invention can still survive and proliferate, and achieve suicide lysis in a normal oxygen concentration environment.

[0151] 4. The bacteria modified by 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 in normal organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figure 1 Agarose gel electrophoresis was used to identify the positive hypoxia promoter clone library for validation.

[0153] (A) Forward hypoxia promoters yhbU-S, tdcA-S, ynfK-S, ynfk-E, tdcA-E, yecH-E, cydA-E, and focA-E;

[0154] (B) Forward hypoxia promoter yhbU-E.

[0155] Figure 2 Agarose gel electrophoresis was used to identify the essential gene library and verify the amplification pattern.

[0156] Figure 3 Agarose gel electrophoresis to identify the reverse high oxygen promoter clone library validation diagram.

[0157] (A) Reverse hyperoxia promoters cyoA-S, phoH-S, lldP-S, argT-S, ydcI-S, phoH-E, lldP-E, ydcI-E;

[0158] (B) Inverted hyperoxia promoter mqo.

[0159] Figure 4 Schematic representation of the hypoxia-specific gene expression cassette.

[0160] Figure 5 Schematic diagram of the lambda RED recombinase and CRE recombinase systems.

[0161] Figure 6 Figure 1. Knockout experiment identification of Salmonella aroA gene.

[0162] (A) Agarose gel electrophoresis to identify the PCR amplification product of the aroA gene knockout fragment;

[0163] (B) Verification of the PCR amplification product of the target fragment of strain SWT003 identified by agarose gel electrophoresis.

[0164] Figure 7 Figure 1. Knockout experiment identification of Salmonella alr.

[0165] (A) Agarose gel electrophoresis to identify the PCR amplification product of the alr gene knockout fragment;

[0166] (B) Verification of the PCR amplification product of the target fragment of strain SWT004 identified by agarose gel electrophoresis.

[0167] Figure 8 Schematic diagram of the construction of plasmid pSWT004 used to construct YB1-like Salmonella SWT005.

[0168] Fig. 9 Agarose gel electrophoresis was used to identify the enzyme digestion products of plasmid pSWT004.

[0169] Fig.10 Verification diagram of PCR amplification product of cm-pepT-asd-sodA fragment identified by agarose gel electrophoresis.

[0170] Fig.11 Verification of PCR amplification products of Salmonella SWT005 identified by agarose gel electrophoresis.

[0171] Fig.12 Figure 2 shows the results of the oxygen adaptability validation test of YB1-like Salmonella SWT005.

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

[0173] (B) Cultivated under aerobic conditions on LB plates without D-alanine.

[0174] Fig.13 Verification of PCR amplification products of plasmid pSWT005 identified by agarose gel electrophoresis.

[0175] Fig.14 Verification of PCR amplification products of strain SWT006 identified by agarose gel electrophoresis.

[0176] Fig.15 Figure 2 shows the results of the oxygen adaptability verification test of the SWT006 strain.

[0177] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0178] (B) Cultivated under aerobic conditions on LB plates without D-alanine.

[0179] Fig.16 Agarose gel electrophoresis identified strain validation library after insertion of the hypoxia-specific gene expression cassette (alr is an essential gene).

[0180] (A)SWT1001, SWT1002, SWT1003, SWT1004, SWT1005, SWT1006, SWT1007, SWT1008;

[0181] (B)SWT1009, SWT1010, SWT1011, SWT1012, SWT1013, SWT1014, SWT1015, SWT1016;

[0182] (C)SWT1017, SWT1018, SWT1019, SWT1020, SWT1021, SWT1022, SWT1023, SWT1024;

[0183] (D)SWT1025, SWT1026, SWT1027, SWT1028, SWT1029, SWT1030, SWT1031, SWT1032;

[0184] (E)SWT1033, SWT1034, SWT1035, SWT1036, SWT1037, SWT1038, SWT1039, SWT1040;

[0185] (F)SWT1041, SWT1042, SWT1043, SWT1044, SWT1045, SWT1046, SWT1047, SWT1048;

[0186] (G)SWT1049, SWT1050, SWT1051, SWT1052, SWT1053, SWT1054, SWT1055, SWT1056;

[0187] (H)SWT1057, SWT1058, SWT1059, SWT1060, SWT1061, SWT1062, SWT1063, SWT1064;

[0188] (I) SWT1065, SWT1066, SWT1067, SWT1068, SWT1069, SWT1070, SWT1071, SWT1072;

[0189] (J)SWT1073, SWT1074, SWT1075.

[0190] Fig.17 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1001.

[0191] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0192] (B) Aerobic culture on LB plates without D-alanine.

[0193] Fig.18Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1002.

[0194] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0195] (B) Aerobic culture on LB plates without D-alanine.

[0196] Fig.19 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1004.

[0197] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0198] (B) Aerobic culture on LB plates without D-alanine.

[0199] Fig. 20 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1005.

[0200] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0201] (B) Aerobic culture on LB plates without D-alanine.

[0202] Fig.21 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1009.

[0203] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0204] (B) Aerobic culture on LB plates without D-alanine.

[0205] Fig. 22 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1010.

[0206] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0207] (B) Aerobic culture on LB plates without D-alanine.

[0208] Fig.23 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1012.

[0209] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0210] (B) Aerobic culture on LB plates without D-alanine.

[0211] Fig.24Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1013.

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

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

[0214] Fig.25 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1021.

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

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

[0217] Fig.26 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1025.

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

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

[0220] Fig. 27 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1033.

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

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

[0223] Fig.28 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1035.

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

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

[0226] Fig.29 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1036.

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

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

[0229] Fig.30Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1038.

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

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

[0232] Fig.31 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1063.

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

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

[0235] Fig.32 Figure 2 shows the oxygen adaptability test results of various Salmonella strains at oxygen concentrations below 0.8%.

[0236] (A) SWT1001 was cultured on LB plates;

[0237] (B) SWT1005 was cultured on LB plates;

[0238] (C) SWT1009 was cultured on LB plates;

[0239] (D) SWT1013 was cultured on LB plates.

[0240] Fig.33 Immunohistochemistry identified the distribution of Salmonella containing a hypoxia-specific gene expression cassette within hypoxic areas of tumors.

[0241] (A) Tumor section after staining with anti-Salmonella antibody. The brown part indicated by the arrow is the antibody-positive area.

[0242] (B) Tumor section stained with anti-HIF1α antibody. The brown part indicated by the arrow is the antibody-positive area.

[0243] Fig.34 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.

[0244] (A) Effect on invasion of CT26 cell line;

[0245] (B) Effect on invasion of EMT6 cell line;

[0246] (C) Effect on invasion of MB49 cell line;

[0247] (D) Invasion effect on RM-1 cell line;

[0248] (E) Invasion effect on A549 cell line;

[0249] (F) Invasion effect on K7M2 cell line;

[0250] (G) Invasion effect on B16F10 cell line;

[0251] (H) Invasion effect on MFC cell line;

[0252] (I) Invasion effect on SCC7 cell line;

[0253] (J) Invasion effect on Hepa1-6 cell line;

[0254] (K) Invasion effect on Pan02 cell line;

[0255] (L) Effect on invasion of ID8 cell line.

[0256] Fig.35 Replenish Fig.34 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.

[0257] (M) Invasion effect on Renca cell line;

[0258] (N) Invasion effect on Neuro-2a cell line.

[0259] Fig.36 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.

[0260] (A) Effect on invasion of CT26 cell line;

[0261] (B) Effect on invasion of EMT6 cell line;

[0262] (C) Effect on invasion of MB49 cell line;

[0263] (D) Invasion effect on RM-1 cell line;

[0264] (E) Invasion effect on A549 cell line;

[0265] (F) Invasion effect on K7M2 cell line;

[0266] (G) Invasion effect on B16F10 cell line;

[0267] (H) Invasion effect on MFC cell line;

[0268] (I) Invasion effect on SCC7 cell line;

[0269] (J) Invasion effect on Hepa1-6 cell line;

[0270] (K) Invasion effect on Pan02 cell line;

[0271] (L) Effect on invasion of ID8 cell line.

[0272] Fig.37 Replenish Fig.36 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.

[0273] (M) Invasion effect on Renca cell line;

[0274] (N) Invasion effect on Neuro-2a cell line.

[0275] Fig.38 The invasion effect of strain SWT1004 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.

[0276] (A) Effect on invasion of CT26 cell line;

[0277] (B) Effect on invasion of EMT6 cell line;

[0278] (C) Effect on invasion of MB49 cell line;

[0279] (D) Invasion effect on RM-1 cell line;

[0280] (E) Invasion effect on A549 cell line;

[0281] (F) Invasion effect on K7M2 cell line;

[0282] (G) Invasion effect on B16F10 cell line;

[0283] (H) Invasion effect on MFC cell line;

[0284] (I) Invasion effect on SCC7 cell line;

[0285] (J) Invasion effect on Hepa1-6 cell line;

[0286] (K) Invasion effect on Pan02 cell line;

[0287] (L) Effect on invasion of ID8 cell line.

[0288] Fig.39 Replenish Fig.38 The invasion effect of strain SWT1004 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.

[0289] (M) Invasion effect on Renca cell line;

[0290] (N) Invasion effect on Neuro-2a cell line.

[0291] Fig.40 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.

[0292] (A) Effect on invasion of CT26 cell line;

[0293] (B) Effect on invasion of EMT6 cell line;

[0294] (C) Effect on invasion of MB49 cell line;

[0295] (D) Invasion effect on RM-1 cell line;

[0296] (E) Invasion effect on A549 cell line;

[0297] (F) Invasion effect on K7M2 cell line;

[0298] (G) Invasion effect on B16F10 cell line;

[0299] (H) Invasion effect on MFC cell line;

[0300] (I) Invasion effect on SCC7 cell line;

[0301] (J) Invasion effect on Hepa1-6 cell line;

[0302] (K) Invasion effect on Pan02 cell line;

[0303] (L) Effect on invasion of ID8 cell line.

[0304] Fig.41 Replenish Fig.40 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.

[0305] (M) Invasion effect on Renca cell line;

[0306] (N) Invasion effect on Neuro-2a cell line.

[0307] Fig.42 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.

[0308] (A) Effect on invasion of CT26 cell line;

[0309] (B) Effect on invasion of EMT6 cell line;

[0310] (C) Effect on invasion of MB49 cell line;

[0311] (D) Invasion effect on RM-1 cell line;

[0312] (E) Invasion effect on A549 cell line;

[0313] (F) Invasion effect on K7M2 cell line;

[0314] (G) Invasion effect on B16F10 cell line;

[0315] (H) Invasion effect on MFC cell line;

[0316] (I) Invasion effect on SCC7 cell line;

[0317] (J) Invasion effect on Hepa1-6 cell line;

[0318] (K) Invasion effect on Pan02 cell line;

[0319] (L) Effect on invasion of ID8 cell line.

[0320] Fig.43 Replenish Fig.42The 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.

[0321] (M) Invasion effect on Renca cell line;

[0322] (N) Invasion effect on Neuro-2a cell line.

[0323] Fig.44 The invasion effect of strain SWT1010 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.

[0324] (A) Effect on invasion of CT26 cell line;

[0325] (B) Effect on invasion of EMT6 cell line;

[0326] (C) Effect on invasion of MB49 cell line;

[0327] (D) Invasion effect on RM-1 cell line;

[0328] (E) Invasion effect on A549 cell line;

[0329] (F) Invasion effect on K7M2 cell line;

[0330] (G) Invasion effect on B16F10 cell line;

[0331] (H) Invasion effect on MFC cell line;

[0332] (I) Invasion effect on SCC7 cell line;

[0333] (J) Invasion effect on Hepa1-6 cell line;

[0334] (K) Invasion effect on Pan02 cell line;

[0335] (L) Effect on invasion of ID8 cell line.

[0336] Fig.45 Replenish Fig.44 The invasion effect of strain SWT1010 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.

[0337] (M) Invasion effect on Renca cell line;

[0338] (N) Invasion effect on Neuro-2a cell line.

[0339] Fig.46 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.

[0340] (A) Effect on invasion of CT26 cell line;

[0341] (B) Effect on invasion of EMT6 cell line;

[0342] (C) Effect on invasion of MB49 cell line;

[0343] (D) Invasion effect on RM-1 cell line;

[0344] (E) Invasion effect on A549 cell line;

[0345] (F) Invasion effect on K7M2 cell line;

[0346] (G) Invasion effect on B16F10 cell line;

[0347] (H) Invasion effect on MFC cell line;

[0348] (I) Invasion effect on SCC7 cell line;

[0349] (J) Invasion effect on Hepa1-6 cell line;

[0350] (K) Invasion effect on Pan02 cell line;

[0351] (L) Effect on invasion of ID8 cell line.

[0352] Fig.47 Replenish Fig.46 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.

[0353] (M) Invasion effect on Renca cell line;

[0354] (N) Invasion effect on Neuro-2a cell line.

[0355] Fig.48The 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.

[0356] (A) Effect on invasion of CT26 cell line;

[0357] (B) Effect on invasion of EMT6 cell line;

[0358] (C) Effect on invasion of MB49 cell line;

[0359] (D) Invasion effect on RM-1 cell line;

[0360] (E) Invasion effect on A549 cell line;

[0361] (F) Invasion effect on K7M2 cell line;

[0362] (G) Invasion effect on B16F10 cell line;

[0363] (H) Invasion effect on MFC cell line;

[0364] (I) Invasion effect on SCC7 cell line;

[0365] (J) Invasion effect on Hepa1-6 cell line;

[0366] (K) Invasion effect on Pan02 cell line;

[0367] (L) Effect on invasion of ID8 cell line.

[0368] Fig.49 Replenish Fig.48 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.

[0369] (M) Invasion effect on Renca cell line;

[0370] (N) Invasion effect on Neuro-2a cell line.

[0371] Fig.50 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.

[0372] (A) Effect on invasion of CT26 cell line;

[0373] (B) Effect on invasion of EMT6 cell line;

[0374] (C) Effect on invasion of MB49 cell line;

[0375] (D) Invasion effect on RM-1 cell line;

[0376] (E) Invasion effect on A549 cell line;

[0377] (F) Invasion effect on K7M2 cell line;

[0378] (G) Invasion effect on B16F10 cell line;

[0379] (H) Invasion effect on MFC cell line;

[0380] (I) Invasion effect on SCC7 cell line;

[0381] (J) Invasion effect on Hepa1-6 cell line;

[0382] (K) Invasion effect on Pan02 cell line;

[0383] (L) Effect on invasion of ID8 cell line.

[0384] Fig.51 Replenish Fig.50 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.

[0385] (M) Invasion effect on Renca cell line;

[0386] (N) Invasion effect on Neuro-2a cell line.

[0387] Fig.52 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.

[0388] (A) Effect on invasion of CT26 cell line;

[0389] (B) Effect on invasion of EMT6 cell line;

[0390] (C) Effect on invasion of MB49 cell line;

[0391] (D) Invasion effect on RM-1 cell line;

[0392] (E) Invasion effect on A549 cell line;

[0393] (F) Invasion effect on K7M2 cell line;

[0394] (G) Invasion effect on B16F10 cell line;

[0395] (H) Invasion effect on MFC cell line;

[0396] (I) Invasion effect on SCC7 cell line;

[0397] (J) Invasion effect on Hepa1-6 cell line;

[0398] (K) Invasion effect on Pan02 cell line;

[0399] (L) Effect on invasion of ID8 cell line.

[0400] Fig.53 Replenish Fig.52 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.

[0401] (M) Invasion effect on Renca cell line;

[0402] (N) Invasion effect on Neuro-2a cell line.

[0403] Fig.54 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.

[0404] (A) Effect on invasion of CT26 cell line;

[0405] (B) Effect on invasion of EMT6 cell line;

[0406] (C) Effect on invasion of MB49 cell line;

[0407] (D) Invasion effect on RM-1 cell line;

[0408] (E) Invasion effect on A549 cell line;

[0409] (F) Invasion effect on K7M2 cell line;

[0410] (G) Invasion effect on B16F10 cell line;

[0411] (H) Invasion effect on MFC cell line;

[0412] (I) Invasion effect on SCC7 cell line;

[0413] (J) Invasion effect on Hepa1-6 cell line;

[0414] (K) Invasion effect on Pan02 cell line;

[0415] (L) Effect on invasion of ID8 cell line.

[0416] Fig.55 Replenish Fig.54 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.

[0417] (M) Invasion effect on Renca cell line;

[0418] (N) Invasion effect on Neuro-2a cell line.

[0419] Fig.56 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.

[0420] (A) Effect on invasion of CT26 cell line;

[0421] (B) Effect on invasion of EMT6 cell line;

[0422] (C) Effect on invasion of MB49 cell line;

[0423] (D) Invasion effect on RM-1 cell line;

[0424] (E) Invasion effect on A549 cell line;

[0425] (F) Invasion effect on K7M2 cell line;

[0426] (G) Invasion effect on B16F10 cell line;

[0427] (H) Invasion effect on MFC cell line;

[0428] (I) Invasion effect on SCC7 cell line;

[0429] (J) Invasion effect on Hepa1-6 cell line;

[0430] (K) Invasion effect on Pan02 cell line;

[0431] (L) Effect on invasion of ID8 cell line.

[0432] Fig.57 Replenish Fig.56 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.

[0433] (M) Invasion effect on Renca cell line;

[0434] (N) Invasion effect on Neuro-2a cell line.

[0435] Fig.58 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.

[0436] (A) Effect on invasion of CT26 cell line;

[0437] (B) Effect on invasion of EMT6 cell line;

[0438] (C) Effect on invasion of MB49 cell line;

[0439] (D) Invasion effect on RM-1 cell line;

[0440] (E) Invasion effect on A549 cell line;

[0441] (F) Invasion effect on K7M2 cell line;

[0442] (G) Invasion effect on B16F10 cell line;

[0443] (H) Invasion effect on MFC cell line;

[0444] (I) Invasion effect on SCC7 cell line;

[0445] (J) Invasion effect on Hepa1-6 cell line;

[0446] (K) Invasion effect on Pan02 cell line;

[0447] (L) Effect on invasion of ID8 cell line.

[0448] Fig.59 Replenish Fig.58The 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.

[0449] (M) Invasion effect on Renca cell line;

[0450] (N) Invasion effect on Neuro-2a cell line.

[0451] Fig.60 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.

[0452] (A) Effect on invasion of CT26 cell line;

[0453] (B) Effect on invasion of EMT6 cell line;

[0454] (C) Effect on invasion of MB49 cell line;

[0455] (D) Invasion effect on RM-1 cell line;

[0456] (E) Invasion effect on A549 cell line;

[0457] (F) Invasion effect on K7M2 cell line;

[0458] (G) Invasion effect on B16F10 cell line;

[0459] (H) Invasion effect on MFC cell line;

[0460] (I) Invasion effect on SCC7 cell line;

[0461] (J) Invasion effect on Hepa1-6 cell line;

[0462] (K) Invasion effect on Pan02 cell line;

[0463] (L) Effect on invasion of ID8 cell line.

[0464] Fig.61 Replenish Fig.60 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.

[0465] (M) Invasion effect on Renca cell line;

[0466] (N) Invasion effect on Neuro-2a cell line.

[0467] Fig.62 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.

[0468] (A) Effect on invasion of CT26 cell line;

[0469] (B) Effect on invasion of EMT6 cell line;

[0470] (C) Effect on invasion of MB49 cell line;

[0471] (D) Invasion effect on RM-1 cell line;

[0472] (E) Invasion effect on A549 cell line;

[0473] (F) Invasion effect on K7M2 cell line;

[0474] (G) Invasion effect on B16F10 cell line;

[0475] (H) Invasion effect on MFC cell line;

[0476] (I) Invasion effect on SCC7 cell line;

[0477] (J) Invasion effect on Hepa1-6 cell line;

[0478] (K) Invasion effect on Pan02 cell line;

[0479] (L) Effect on invasion of ID8 cell line.

[0480] Fig.63 Replenish Fig.62 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.

[0481] (M) Invasion effect on Renca cell line;

[0482] (N) Invasion effect on Neuro-2a cell line.

[0483] Fig.64Comparison of 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.

[0484] (A) Comparison of the killing effects of strain SWT005 and strain SWT1001 on different cancer cell lines;

[0485] (B) Comparison of the killing effects of strains SWT005 and SWT1002 on different cancer cell lines.

[0486] Fig.65 Comparison of 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.

[0487] (A) Comparison of the killing effects of strain SWT005 and strain SWT1004 on different cancer cell lines;

[0488] (B) Comparison of the killing effects of strains SWT005 and SWT1005 on different cancer cell lines.

[0489] Fig.66 Comparison of 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.

[0490] (A) Comparison of the killing effects of strain SWT005 and strain SWT1009 on different cancer cell lines;

[0491] (B) Comparison of the killing effects of strain SWT005 and strain SWT1010 on different cancer cell lines.

[0492] Fig.67 Comparison of 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.

[0493] (A) Comparison of the killing effects of strain SWT005 and strain SWT1012 on different cancer cell lines;

[0494] (B) Comparison of the killing effects of strain SWT005 and strain SWT1013 on different cancer cell lines.

[0495] Fig.68 Comparison of 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.

[0496] (A) Comparison of the killing effects of strain SWT005 and strain SWT1021 on different cancer cell lines;

[0497] (B) Comparison of the killing effects of strains SWT005 and SWT1025 on different cancer cell lines.

[0498] Fig.69 Comparison of 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.

[0499] (A) Comparison of the killing effects of strain SWT005 and strain SWT1033 on different cancer cell lines;

[0500] (B) Comparison of the killing effects of strains SWT005 and SWT1035 on different cancer cell lines.

[0501] Fig.70 Comparison of 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.

[0502] (A) Comparison of the killing effects of strain SWT005 and strain SWT1036 on different cancer cell lines;

[0503] (B) Comparison of the killing effects of strain SWT005 and strain SWT1038 on different cancer cell lines.

[0504] Fig.71 Comparison of 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.

[0505] Fig.72 Figure 3. Targeting evaluation of strain SWT005 in mouse tumor models.

[0506] Bacterial counts in the Heart, Liver, Spleen, Lung, Kidney, and Tumor on the 1st day (D1), 5th day (D5), and 11th day (D11) after mice were injected with SWT005 via the tail vein.

[0507] Fig.73 Targeting evaluation diagram of strain SWT1001, strain SWT1002, strain SWT1004, and strain SWT1005 in mouse tumor models.

[0508] The bacterial counts in the Heart, Liver, Spleen, Lung, Kidney, and Tumor were measured on the 1st day (D1), 5th day (D5), and 11th day (D11) after the mice were injected with Salmonella strains via the tail vein. The statistical analysis was compared with the strain SWT005.

[0509] (A) Strain SWT1001;

[0510] (B) Strain SWT1002;

[0511] (C) Strain SWT1004;

[0512] (D) Strain SWT1005.

[0513] Fig.74 Targeting evaluation diagram of strain SWT1009, strain SWT1010, strain SWT1012, and strain SWT1013 in mouse tumor models.

[0514] The bacterial counts in the Heart, Liver, Spleen, Lung, Kidney, and Tumor were measured on the 1st day (D1), 5th day (D5), and 11th day (D11) after the mice were injected with Salmonella strains via the tail vein. The statistical analysis was compared with the strain SWT005.

[0515] (A) Strain SWT1009;

[0516] (B) Strain SWT1010;

[0517] (C) Strain SWT1012;

[0518] (D) Strain SWT1013.

[0519] Fig.75 Targeting evaluation diagram of strain SWT1021, strain SWT1025, strain SWT1033, and strain SWT1035 in mouse tumor models.

[0520] The bacterial counts in the Heart, Liver, Spleen, Lung, Kidney, and Tumor were measured on the 1st day (D1), 5th day (D5), and 11th day (D11) after the mice were injected with Salmonella strains via the tail vein. The statistical analysis was compared with the strain SWT005.

[0521] (A) Strain SWT1021;

[0522] (B) strain SWT1025;

[0523] (C) Strain SWT1033;

[0524] (D) Strain SWT1035.

[0525] Fig.76 Targeting evaluation diagram of strains SWT1036, SWT1038, and SWT1063 in mouse tumor models.

[0526] The bacterial counts in the Heart, Liver, Spleen, Lung, Kidney, and Tumor were measured on the 1st day (D1), 5th day (D5), and 11th day (D11) after the mice were injected with Salmonella strains via the tail vein. The statistical analysis was compared with the strain SWT005.

[0527] (A) strain SWT1036;

[0528] (B) strain SWT1038;

[0529] (C) Strain SWT1063.

[0530] Fig.77The tumor inhibitory effects of strain SWT005 were compared with those of strain SWT1001, strain SWT1002, strain SWT1004, and strain SWT1005 in mouse tumor models, with Vehicle serving as the control group.

[0531] (A) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1001 in mouse tumor models;

[0532] (B) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1002 in mouse tumor models;

[0533] (C) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1004 in mouse tumor models;

[0534] (D) Comparison of the tumor inhibitory effects of strains SWT005 and SWT1005 in mouse tumor models.

[0535] Fig.78 The tumor inhibitory effects of strain SWT005 were compared with those of strain SWT1009, strain SWT1010, strain SWT1012, and strain SWT1013 in mouse tumor models, with Vehicle serving as the control group.

[0536] (A) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1009 in mouse tumor models;

[0537] (B) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1010 in mouse tumor models;

[0538] (C) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1012 in mouse tumor models;

[0539] (D) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1013 in mouse tumor models.

[0540] Fig.79 The tumor inhibitory effects of strain SWT005, strain SWT1021, strain SWT1025, strain SWT1033, and strain SWT1035 in mouse tumor models were compared, with Vehicle serving as the control group.

[0541] (A) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1021 in mouse tumor models;

[0542] (B) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1025 in mouse tumor models;

[0543] (C) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1033 in mouse tumor models;

[0544] (D) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1035 in mouse tumor models.

[0545] Fig.80 Comparison of the tumor inhibitory effects of strain SWT005, strain SWT1036, strain SWT1038, and strain SWT1063 in mouse tumor models, with Vehicle serving as the control group.

[0546] (A) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1036 in mouse tumor models;

[0547] (B) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1038 in mouse tumor models;

[0548] (C) Comparison of the tumor inhibitory effects of strain SWT005 and strain SWT1063 in mouse tumor models.

[0549] Fig.81 This is a verification diagram of the PCR amplification product of SWT007 identified by agarose gel electrophoresis.

[0550] Fig.82 Agarose gel electrophoresis was used to identify the strain validation library after the insertion of the hypoxia-specific gene expression cassette (dadX is an essential gene).

[0551] (A)SWT2001, SWT2002, SWT2004, SWT2005;

[0552] (B)SWT2009, SWT2010, SWT2012, SWT2013;

[0553] (C)SWT2033, SWT2035, SWT2036, SWT2038;

[0554] (D) SWT2021, SWT2025;

[0555] (E)SWT2063.

[0556] Fig.83 Graph showing the oxygen adaptability of strains SWT2001, SWT2002, SWT2004, and SWT2005.

[0557] (A) SWT2001 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine.

[0558] (B) SWT2002 was cultured on an LB plate without D-alanine, with the upper plate being cultured under anaerobic conditions and the lower plate being cultured under aerobic conditions;

[0559] (C) SWT2004 was cultured on an LB plate without D-alanine, with the upper plate being cultured under anaerobic conditions and the lower plate being cultured under aerobic conditions;

[0560] (D) SWT2005 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine.

[0561] Fig.84 Validation diagram of oxygen adaptability of strains SWT2009, SWT2010, SWT2012, and SWT2013.

[0562] (A) SWT2009 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine.

[0563] (B) SWT2010 was cultured on an LB plate without D-alanine, with the upper plate cultured under anaerobic conditions and the lower plate cultured under aerobic conditions;

[0564] (C) SWT2012 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic culture and the lower plate showing aerobic culture.

[0565] (D) SWT2013 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine.

[0566] Fig.85 Validation diagram of oxygen adaptability of strains SWT2033, SWT2035, SWT2036, and SWT2038.

[0567] (A) SWT2033 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine.

[0568] (B) SWT2035 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic culture and the lower plate showing aerobic culture.

[0569] (C) SWT2036 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic culture and the lower plate showing aerobic culture.

[0570] (D) SWT2038 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine.

[0571] Fig.86Validation diagram of oxygen adaptability of strains SWT2021, SWT2025 and SWT2063.

[0572] (A) SWT2021 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine;

[0573] (B) SWT2025 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic culture and the lower plate showing aerobic culture.

[0574] (C) SWT2063 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine.

[0575] Fig.87 Validation diagram of oxygen adaptability of strains SWT1065, SWT1066, SWT1068, and SWT1069.

[0576] (A) SWT1065 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine.

[0577] (B) SWT1066 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine;

[0578] (C) SWT1068 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic culture and the lower plate showing aerobic culture.

[0579] (D) SWT1069 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine.

[0580] Fig.88 Oxygen adaptability validation diagram of strains SWT1041, SWT1042, SWT1044, and SWT1045.

[0581] (A) SWT1041 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine.

[0582] (B) SWT1042 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine;

[0583] (C) SWT1044 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine;

[0584] (D) SWT1045 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine.

[0585] Fig.89 Validation diagram of oxygen adaptability of strains SWT1008, SWT1016, and SWT1024.

[0586] (A) SWT1008 was cultured on an LB plate without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0587] (B) SWT1016 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine;

[0588] (C) SWT1024 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on LB plates without D-alanine.

[0589] Fig.90 Verification of oxygen adaptability of strains SWT1073, SWT1074, and SWT1075.

[0590] (A) SWT1073 was cultured under anaerobic conditions on LB plates without D-alanine.

[0591] The following is aerobic culture;

[0592] (B) SWT1074 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine;

[0593] (C) SWT1075 was cultured under anaerobic conditions on the upper side and aerobic conditions on the lower side on an LB plate without D-alanine. DETAILED DESCRIPTION

[0594] The present application is further described in detail below through the accompanying drawings and embodiments. Through these descriptions, the characteristics and advantages of the present application will become clearer and more specific.

[0595] Example 1: Construction of a hypoxia-specific gene expression cassette with the alr gene as a survival essential gene

[0596] In this embodiment, the forward hypoxia promoters are respectively 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), 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 high oxygen promoters are respectively Salmonella cyoA (cyoA-S) (SEQ ID No. 19). 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), Escherichia coli mqo (SEQ ID No.62), to construct a hypoxia-specific gene expression cassette.

[0597] The combination of hypoxia-specific gene expression cassettes is shown in Table 1.

[0598] Table 1 Combination table of forward hypoxia promoter and reverse hyperoxia promoter (where the essential survival gene is the alr gene of Salmonella)

[0599]

[0600] (I) Construction of positive hypoxia promoter clone library

[0601] 1. Pick a single clone 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 culture medium, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours. OD 600 The reading is between 2-3;

[0602] 2. Use a pipette to absorb 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 mix and put it into the PCR amplification equipment.

[0603] 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;

[0604] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0605] 4. Mix the recovered product with the endonuclease (purchased from NEB) digestion system and incubate at 37°C for 1 hour;

[0606] 5. Purify and recover the enzyme cleavage products through a DNA gel recovery system.

[0607] The primers and endonucleases used for different forward hypoxia promoters are as follows:

[0608] 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;

[0609] 2. ynfK-E (SEQ ID No. 2): amplified from E. coli strain DH10B using primers SWTO21, 22, and the product was double-cut with NotI and HindIII and recovered;

[0610] 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;

[0611] 4. tdcA-E (SEQ ID No. 4): amplified from E. coli strain DH10B using primers SWTO25, 26, and the product was double-cut with NotI and HindIII and recovered;

[0612] 5. yecH-E (SEQ ID No. 5): amplified by primers SWTO27, 28 from Escherichia coli strain DH10B, and the product was double-cut with NotI and HindIII and recovered;

[0613] 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;

[0614] 7. cydA-E (SEQ ID No. 7): amplified from E. coli strain DH10B using primers SWTO31, 32, and the product was double-cut with NotI and HindIII and recovered;

[0615] 8. focA-E (SEQ ID No. 8): The E. coli strain DH10B was amplified using primers SWTO33, 34, and the product was double-cut with NotI and HindIII and recovered.

[0616] 9. yhbU-E (SEQ ID No. 48): The E. coli strain DH10B was amplified using primers SWTO82, 83, and the product was double-cut with NotI and HindIII and recovered.

[0617] The results of the verification of the positive hypoxia promoter clone library by agarose gel electrophoresis are shown in the attached figure. Figure 1 (A) and Figure 1 (B) shown.

[0618] (II) Construction of a library of genes essential for survival

[0619] 1. Pick a single clone 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 culture medium, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0620] 2. Use a pipette to absorb 2 μl of the corresponding strain culture medium and mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then mix and place it in the PCR amplification device.

[0621] 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;

[0622] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0623] 4. Mix the recovered product with the enzyme digestion system and incubate at 37 degrees for 1 hour;

[0624] 5. Purify and recover the enzyme cleavage products through a DNA gel recovery system.

[0625] The genes essential for survival from different sources are as follows:

[0626] 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;

[0627] 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;

[0628] 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;

[0629] 4. Escherichia coli dadX gene (SEQ ID No. 23): The strain DH10B was amplified using primers SWTO41 and 42, and the product was double-cut with HindIII and XhoI and recovered.

[0630] The results of the verification of the survival essential gene library by agarose gel electrophoresis are shown in the attached Figure 2 shown.

[0631] (III) Construction of the reverse hyperoxia promoter library

[0632] 1. Pick a single clone 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 culture medium, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0633] 2. Use a pipette to absorb 2 μl of the corresponding strain culture medium and mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then mix and place it in the PCR amplification device.

[0634] 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;

[0635] 3. The amplified PCR product is purified and recovered through a DNA gel recovery system;

[0636] 4. Mix the recovered product with the enzyme digestion system and incubate at 37°C for 1 hour;

[0637] 5. Purify and recover the enzyme cleavage products through a DNA gel recovery system.

[0638] The primers and endonucleases used for different reverse hyperoxia promoters are as follows:

[0639] 1. cyoA-S (SEQ ID No. 9): strain SWT001 was amplified using primers SWTO43 and 44, and the product was double-cut with XhoI and PstI and recovered;

[0640] 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;

[0641] 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;

[0642] 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;

[0643] 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;

[0644] 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;

[0645] 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;

[0646] 8. ydcI-E (SEQ ID No. 16): The strain DH10B was amplified using primers SWTO57 and 58, and the product was double-cut with XhoI and PstI and recovered.

[0647] 9. mqo (SEQ ID No. 62): The strain DH10B was amplified using primers SWTO7 and 8, and the product was double-cut with XhoI and PstI and recovered.

[0648] The results of the verification of the reverse high oxygen promoter clone library by agarose gel electrophoresis are shown in the attached figure. Figure 3 (A) and Figure 3 (B) shown.

[0649] (IV) Construction of a combinatorial library of positive hypoxia promoters, survival essential genes, and negative hyperoxia promoters

[0650] The composition of the hypoxia-specific gene expression cassette is shown in the attached Figure 4 As shown, it is composed of a positive hypoxia promoter, a survival essential gene and a negative hyperoxia promoter in sequence.

[0651] As shown in Table 1, a cloned library was formed by combining the sequences of the forward hypoxia promoter library digested by NotI and HindIII, the survival essential gene Salmonella alr gene (SEQ ID No. 17) digested by HindIII and XhoI, and the reverse hyperoxia promoter library digested by XhoI and PstI, and then connected into the plasmid pSWT003 vector (vector backbone, pBlueScript SK (+), purchased from Biowind) digested by SpeI and PstI, and the plasmid pSWT007 vector (containing the double-sided same-direction loxp sequences and the DNA fragment of the chloramphenicol resistance gene (SEQ ID No. 61)) digested by SpeI and NotI.

[0652] The products of Salmonella SWT001 or Escherichia coli DH10B amplified by the 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.

[0653] The construction of each strain of the present invention is as follows:

[0654] 1. Wild-type Salmonella typhimurium (SWT001)

[0655] Wild-type Salmonella typhimurium (SWT001) was purchased from China Industrial Microbiology Culture Collection Center (CICC).

[0656] 2. Construction of Salmonella (SWT002) containing temperature-induced lambda-RED recombinase and loxp-CRE enzyme system

[0657] The Lambda-RED recombination system is widely used in homologous recombination of Gram-negative bacteria. In the present invention, this system is composed of plasmid pSWT001. Plasmid pSWT001 is as shown in the attached Figure 5As shown, it includes a lambda-RED recombinase module (SEQID No.60) (similar in function to the plasmid vector psim6 of Biowind) and a loxp-Cre recombinase module (SEQ IDNo.60) (similar in function to the plasmid vector 705-Cre of Gene Bridges). The lambda-RED recombinase module consists of three recombinases, EXO, BET, and GAM, and the recombinase is controlled by the CI857 temperature control regulator. Therefore, it cannot be expressed under the culture condition of 32°C, and it will only be expressed when it is higher than 37°C. Under temperature-induced conditions, the recombinase EXO cuts the 5' end of the double-stranded linear DNA to cause a single-stranded DNA protruding at the 3' end. The single-stranded DNA will be bound by the BET protein and protected from degradation by other nucleases. The role of GAM is to inhibit the action of endogenous nucleases in bacteria. The homologous arms of homologous recombination are about 35-50bp, which are added on both sides of the DNA fragment to be recombined by 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 homology arm sequences of the positions to be recombined, and inside are loxp sequences in the same direction on both sides, totaling 34bp. The sites of the double loxp in the same direction are chloramphenicol resistance genes, which are used to screen recombinant bacteria. After successful recombination, the CRE enzyme system carried on the pSWT001 plasmid (also controlled by the CI857 temperature control regulator) specifically recognizes the loxp sequence, and can cut the sequence in the middle of the loxp in the same direction, leaving a loxp sequence, thereby eliminating the chloramphenicol resistance gene. Repeating the above operation can achieve continuous knockout and knock-in of genes.

[0658] The specific steps are as follows:

[0659] (1) Streak strain SWT001 on an LB plate and culture it statically in a 37°C incubator overnight;

[0660] (2) Pick a single clone and inoculate it into 5 ml of LB liquid culture medium, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0661] (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 grows to OD 600 =0.3 Place on ice and let stand for 1 hour;

[0662] (4) Wash the cells three times with sterilized pure water;

[0663] (5) The recovered cells were mixed with 10 ng of plasmid pSWT001 and electroporated at a voltage of 1.8 kV;

[0664] (6) The electroporated cells were spread on LB plates containing 100 μg / ml ampicillin sodium and cultured in a 32°C incubator overnight until a single clone colony was grown, which was named SWT002.

[0665] 3. Construction of attenuated Salmonella with aroA gene knockout (SWT003)

[0666] (1) A single clone of SWT002 was selected 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.

[0667] (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 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.

[0668] (3) Wash the cells three times with sterilized pure water.

[0669] (4) Preparation of PCR products of SWTO1 and SWTO2.

[0670] 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.

[0671] 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.

[0672] The amplified PCR products were verified by agarose gel electrophoresis for the PCR amplification products of SWTO1 and SWTO2 of the aroA gene knockout fragment. The verification results are shown in the attached Figure 6 As shown in A.

[0673] The PCR products were purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR products were measured by nanodrop.

[0674] (5) The recovered bacteria were mixed with 100 ng of PCR products of SWTO1 and SWTO2 and electroporated at a voltage of 1.8 kV.

[0675] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured in a 32°C incubator overnight until a single clone colony was grown.

[0676] (7) Positive clones were identified by colony PCR, and the insertion of chloramphenicol resistance gene was identified by using SWTO4, SWTO6, SWTO3 and SWTO5.

[0677] The target fragment PCR amplification product of strain SWT003 was verified by agarose gel electrophoresis. The verification results are shown in the attached Figure 6 As shown in B.

[0678] (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.

[0679] 4. Construction of alr gene knockout attenuated bacteria (SWT004)

[0680] (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.

[0681] (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 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.

[0682] (3) Wash the cells three times with sterilized pure water.

[0683] (4) Preparation of PCR products of SWTO70 and SWTO71.

[0684] Primers SWTO70 and SWTO71 were mixed with plasmid pSWT002 and a high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0685] 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.

[0686] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0687] (5) The recovered bacteria were mixed with 100 ng of PCR products of SWTO70 and SWTO71 and electroporated at a voltage of 1.8 kV.

[0688] 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 the attached Figure 7 As shown in A.

[0689] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured in a 32°C incubator overnight until a single clone colony was grown.

[0690] (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.

[0691] The target fragment PCR amplification product of strain SWT004 was verified by agarose gel electrophoresis. The verification results are shown in the attached Figure 7 As shown in B (the primers used are SWTO72, SWTO5, SWTO6, and SWTO73).

[0692] (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.

[0693] 5. Construction of YB1-like Salmonella (SWT005)

[0694] In order to study a more preferred oxygen-regulated promoter combination, the present invention constructed the YB1 Salmonella strain recorded in Chinese patent CN104471057B as a prior art strain for comparison with the strain constructed by the present invention.

[0695] The YB1-like Salmonella strain includes aroA gene defect and an oxygen regulation system constructed by the forward hypoxic promoter pepT, the essential gene asd and the reverse hyperoxic promoter sodA.

[0696] (1) Construction of plasmid pSWT004

[0697] ①Construct the pSWT004 plasmid as shown in the attached Figure 8 As shown, there are chloramphenicol resistance gene cm (SEQ ID No.43) with loxp sequences on both sides, pepT promoter (SEQ ID No.44), asd gene (SEQ ID No.45), and reverse sodA promoter (SEQ ID No.47).

[0698] ② Plasmid pSWT002 was digested with NotI and a 1130 bp fragment was recovered. Strain SWT001 was amplified with primers SWTO11 and SWTO12, and the product was double-cut with NotI and HindIII and recovered.

[0699] The strain SWT001 was amplified by primers SWTO13 and SWTO14, and the product was double-cut with XhoI and HindIII and recovered.

[0700] The primers SWTO15 and SWTO16 were directly annealed to generate XhoI and PstI nicks, and then recovered.

[0701] ③ Double-cut the plasmid pSWT003 (plasmid backbone) with NotI and PstI, connect the five fragments with T4 DNA ligase, spread on ampicillin and chloramphenicol double-resistant LB plates, and screen single clones, named pSWT004 (YB1-like construction plasmid, including cm-pepT-asd-sodA).

[0702] ④Extract plasmid pSWT004, take 100 ng and mix with SacI and KpnI endonuclease system, incubate at 37℃ for 1 hour, and identify by agarose gel electrophoresis. Fig. 9 shown.

[0703] (2) Construction of recombinant YB1-like Salmonella SWT005 (YB1-like)

[0704] Plasmid pSWT004 was amplified and recovered using primers SWTO17 and SWTO18, and this fragment was recombined into SWT003 to construct YB1-like Salmonella SWT005.

[0705] ① Pick a single clone of SWT003 and inoculate it into 5 ml LB liquid culture medium, and place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0706] ②Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing 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.

[0707] ③ Wash the bacteria 3 times with sterilized pure water and set aside.

[0708] ④ Amplify pSWT004 with primers SWTO17 and SWTO18, purify and recover the recovered PCR products. The PCR amplification products of cm-pepT-asd-sodA fragments SWTO17 and SWTO18 were verified by agarose gel electrophoresis. The verification results are shown in the attached figure. Fig.10 As shown, the concentration and purity of DNA were determined by nanodrop.

[0709] ⑤ Mix the recovered bacteria with 100 ng of PCR product and perform electroporation at a voltage of 1.8 kV.

[0710] 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 constant temperature incubator at 32°C overnight until a single clone colony grows.

[0711] ⑦ The positive clones were identified by colony PCR, and the insertion of chloramphenicol resistance gene was identified by SWTO93, SWTO5, SWTO94 and SWTO95. The results of the verification of PCR amplification products of Salmonella SWT005 by agarose gel electrophoresis (SWTO93 and SWTO5, SWTO94 and SWTO95) are shown in the attached figure. Fig.11 shown.

[0712] (3) Verification of oxygen adaptability of YB1-like Salmonella SWT005

[0713] 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.

[0714] ②After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0715] ③ Calculate the volume of bacterial solution (microliters) per 1OD of bacteria according to the following formula, and add deionized water to 1ml.

[0716]

[0717] ④ Take 10 μl of the above bacterial solution and spot it on two LB plates without DAP for culture, marked as "1", and repeat three times, marked as "a", "b", and "c".

[0718] ⑤ After 10-fold gradient dilution, take another 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0719] ⑥ Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0720] ⑦ One of the plates was placed in an anaerobic tank at 37°C. The operation process was to place the anaerobic gas-producing bag (brand: Japan Mitsubishi, item number: D-119) combination into a 7.0L sealed culture tank (brand: Japan Mitsubishi, item 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.

[0721] The results of the oxygen adaptability verification test of YB1-like Salmonella SWT005 are shown in the attached Fig.12 shown.

[0722] 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 LB plates without DAP (such as attached Fig.12 (A)), under aerobic (atmospheric environment) conditions 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 strains (such as Fig.12 (as shown in (B)).

[0723] 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, and further indicates that the oxygen regulation system of YB1 Salmonella SWT005 has certain defects.

[0724] 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 is more inclined to target the hypoxic area of ​​the tumor.

[0725] 6. Verification of essential gene replacement and construction of SWT006 strain

[0726] 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.

[0727] Therefore, the present invention replaces the essential gene asd in the oxygen regulation expression cassette of YB1-like Salmonella SWT005 with the alr gene, creates a new hypoxia-specific regulation expression cassette (pepT-alr-sodA), and verifies its oxygen regulation function.

[0728] Since double mutations of the alr and dadX genes will cause lethal traits, it is necessary to first construct the SWT004 strain (a strain with aroA gene knockout and alr gene knockout), and then integrate the hypoxia-specific regulatory expression cassette (pepT-alr-sodA) into the dadX gene site of the SWT004 strain (to destroy its function) to construct the SWT006 strain.

[0729] (1) Construction of plasmid pSWT005

[0730] Based on SWT004, a strain with pepT promoter, alr as an essential gene, and a hypoxia-specific gene expression cassette of the reverse sodA promoter was integrated into the dadX gene locus of SWT004. The construction process is as follows:

[0731] ① Cut the plasmid pSWT004 (YB1-like plasmid, including cm-pepT-asd-sodA) with HindIII and XhoI, cut out the asd fragment, and recover the remaining fragments.

[0732] ② The strain SWT001 was amplified using primers SWTO35 and SWTO36 to obtain the amplified product of the alr gene of Salmonella, which was double-cut with HindIII and XhoI and recovered.

[0733] ③ Connect the above fragments using T4 DNA ligase.

[0734] ④ Transform into DH10B competent cells, spread on LB plates containing ampicillin and chloramphenicol, and culture inverted at 37°C for 16 hours.

[0735] ⑤ Pick a single clone and perform PCR identification using primers SWTO11 and SWTO36. Verify the PCR amplification products of SWTO11 and SWTO36 by agarose gel electrophoresis. The verification results are shown in the attached Fig.13 shown.

[0736] ⑥ Shake the bacteria, extract the plasmid, and obtain plasmid pSWT005 (cm-pepT-alr-sodA element).

[0737] (2) Hypoxia-specific gene expression cassette pepT-alr-sodA strain (SWT006, aroA - &alr - Construction of pepT-alr-sodA

[0738] ① Pick a single clone of the alr gene knockout attenuated bacteria SWT004 and inoculate it into 5 ml LB liquid culture medium, and place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0739] ②Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing 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.

[0740] ③ Wash the bacteria 3 times with sterilized pure water and set aside.

[0741] ④ Amplify pSWT005 (cm-pepT-alr-sodA) using primers SWTO89 and SWTO90, purify and recover the PCR product, and determine the DNA concentration and purity using nanodrop.

[0742] ⑤ Mix the recovered bacteria with 100 ng of PCR product and perform electroporation at a voltage of 1.8 kV.

[0743] ⑥ Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and culture them in a constant temperature incubator at 32°C overnight until a single clone colony grows.

[0744] ⑦ The positive clones were identified by colony PCR. SWTO87, SWTO76, SWTO59 and SWTO77 were used to identify the insertion of chloramphenicol resistance gene. The PCR amplification product of Salmonella SWT006 was verified by agarose gel electrophoresis. The verification results are shown in the attached figure. Fig.14 shown.

[0745] (3) Verification of oxygen adaptability of SWT006 strain

[0746] ① Pick a single clone of the SWT006 strain and inoculate it into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0747] ②After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0748] ③ Calculate the volume of bacterial solution per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0749]

[0750] 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".

[0751] ⑤ After 10-fold gradient dilution, take another 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0752] ⑥ Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0753] ⑦ One of the plates was cultured in an anaerobic environment at 37°C (oxygen concentration was less than 0.01%), and the other plate was cultured in an atmospheric environment at 37°C (21% oxygen concentration).

[0754] The results of the oxygen adaptability test of SWT006 strain are shown in the attached Fig.15 As shown in (A), the SWT006 strain can grow normally in an anaerobic environment.

[0755] 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 SWT005 YB1-like, the selection of alr as an essential gene is better regulated than the asd gene of SWT005 YB1-like under aerobic conditions, as shown in the attached Fig.15 As shown in (B), only at concentration 1 (10 -2 OD), concentration 2(10 -3 OD) and concentration 3(10 -4 OD) showed obvious growth of the strain.

[0756] 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.

[0757] Example 2: Integrating the hypoxia-specific gene expression cassette into the dadX gene locus on the chromosome of the SWT004 strain (destroying its function)

[0758] (I) Construction of alr-deficient attenuated bacteria containing a hypoxia-specific gene expression cassette

[0759] 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, and place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0760] 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 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.

[0761] 3. Wash the bacteria 3 times with sterilized pure water.

[0762] 4. Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette, mix primers SWTO78 and SWTO79 with a hypoxia-specific gene expression cassette plasmid, and a high-fidelity PCR amplification enzyme system and place them into a PCR amplification device.

[0763] 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.

[0764] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0765] 5. Mix the recovered bacteria with 100 ng of PCR product and perform electroporation at a voltage of 1.8 kV.

[0766] 6. Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and culture them in a constant temperature incubator at 32°C overnight until a single clone colony grows.

[0767] 7. Inoculate the single clone into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and culture it 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.

[0768] 8. Use the corresponding primers to identify the clones by colony PCR. The identification results are shown in the attached Fig.16 shown.

[0769] 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 .

[0770] Table 2 Strain construction table

[0771]

[0772]

[0773]

[0774]

[0775]

[0776]

[0777] Table 3 Correspondence between strains and identification primers

[0778]

[0779]

[0780]

[0781] (II) Oxygen adaptability verification of the Salmonella strain library containing the hypoxia-specific gene expression cassette

[0782] 1. Pick a single clone of the strain containing the hypoxia-specific expression cassette and inoculate it into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0783] 2. After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0784] 3. Calculate the volume of bacterial solution per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0785]

[0786] 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".

[0787] 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".

[0788] 6. Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0789] 7. One of the plates 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).

[0790] The results of the oxygen adaptability verification test of the strain containing the hypoxia specific expression cassette are shown in the attached Figure 17-31 shown.

[0791] Based on the data of the aforementioned YB1-like (SWT005) and the data of 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 show 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 evaluation.

[0792] (III) Oxygen concentration simulation of strain library containing hypoxia-specific gene expression cassettes

[0793] By using anaerobic gas production bags to consume 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:

[0794] The anaerobic gas production bag (brand: Japan Mitsubishi, item number: D-119) was combined and placed in a 7.0L sealed culture tank (brand: Japan Mitsubishi, item number: D-112), and an oxygen meter (brand: Meicheng Electrochemical, item number: OX-100A) was also placed in the tank.

[0795] The above anaerobic gas-generating bag combination was used to verify the growth of the genetically modified strains below 0.8% oxygen concentration.

[0796] 1. Verification of the adaptability of the strain library containing hypoxia-specific gene expression cassettes to oxygen concentrations below 0.8%

[0797] Since the pathological hypoxic zone of a tumor has an oxygen concentration below 1%, the present invention intends to prove that when the oxygen concentration is below 1% (or close to 1%), the Salmonella transformed with the hypoxia-specific gene expression cassette can still grow normally.

[0798] The oxygen concentration is regulated by the anaerobic gas production bag. After the anaerobic gas production bag is consumed for 1 hour, the oxygen meter reading shows that the oxygen concentration is 0.8-1%. Therefore, this experiment simulates the hypoxic environment inside the tumor, verifies the growth of Salmonella on the culture dish after the anaerobic gas production bag consumes the sealed culture tank for 1 hour, and simulates the growth of Salmonella strains containing hypoxia-specific gene expression boxes in the hypoxic environment inside the tumor.

[0799] The operation process of the spot plate experiment is as follows:

[0800] (1) A single clone of the strain containing the hypoxia-specific expression cassette was selected and inoculated into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and placed in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0801] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0802] (3) Calculate the volume of bacterial solution per 1 OD of bacteria according to the following formula and add deionized water to 1 ml.

[0803]

[0804] (4) Take 10 μl of the above bacterial solution and spot it on an LB plate containing D-alanine for culture, label it as “1” and repeat three times, label them as “a”, “b” and “c”.

[0805] (5) At the same time, take 10 μl of the above bacterial solution and spot it on another LB plate without D-alanine for culture, mark it as "1", and spot three replicates, mark them as "a", "b", and "c".

[0806] (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”.

[0807] (7) Repeat this 10-fold serial dilution until the mark “8” is reached.

[0808] (8) The two plates were incubated at 37°C in an environment of 0.8% oxygen.

[0809] As attached Fig.32 As shown, the present invention compares different strains (attached Fig.32 (A) SWT1001, Attachment Fig.32 (B) STW1005, Attachment Fig.32 (C) SWT1009, Attachment Fig.32 (D) SWT1013) was measured on LB plates without D-alanine to determine the growth of each strain below 0.8% oxygen concentration. Since the culture medium without D-alanine was used, the corresponding Salmonella strains needed to rely on their own oxygen regulation system to grow normally. The results confirmed that strains containing low oxygen specific expression cassettes that can grow normally in an anaerobic environment can all grow normally below 0.8% oxygen concentration.

[0810] 2. Immunohistochemical staining of hypoxia-specific gene expression cassette strain (SWT1005) and tumor hypoxia area

[0811] To demonstrate the accumulation of Salmonella containing a hypoxia-specific gene expression cassette in hypoxic areas of tumors, mouse tumor models injected with Salmonella via 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.

[0812] (1) Establishment of tumor model

[0813] BALB / c mice (purchased from Zhejiang Weitong Lihua 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 100mm 3 The mice were divided into PBS group and SWT1005 group, with 5 mice in each group. The tumor-bearing mice were inoculated with 1×10 7 CFU bacteria.

[0814] (2) Bacterial preparation

[0815] Taking strain SWT1005 as an example, streak on an LB plate to which D-alanine was additionally added, and cultured statically in a 37°C constant temperature incubator overnight; pick a single clone of strain SWT1005 and inoculate it into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours; measure the absorbance (OD value) of the strain at 600 nm, calculate and measure the culture with an OD value of 1, wash it three times with PBS, and prepare a bacterial suspension in PBS.

[0816] (3) Tumor slice preparation

[0817] 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 animals were embedded in paraffin. After complete cooling and solidification, the slices were sliced ​​and dewaxed. The slices were immersed in sodium citrate antigen repair solution, boiled for 10 minutes for antigen repair, and then rinsed with PBS three times. The antigen-recovered slices were immersed in 3% hydrogen peroxide solution for 10 minutes, and then rinsed with PBS three times. The slices after catalase removal were immersed in 2% BSA-PBS solution, blocked at room temperature for 1 hour, and then rinsed with PBS three times. Anti-Salmone The primary antibody incubation solution of lla (purchased from abcam, catalog number ab35156) and anti-HIF1α (purchased from abcam, catalog number ab51608) was added to the different blocked tissue sections, incubated at 4°C overnight, and then rinsed with PBS three times; the secondary antibody incubation solution was added to the tissue sections, incubated for 25 minutes, and then rinsed with PBS three times; DAB staining solution was added to the tissue sections, and hematoxylin was used for counterstaining for 3 minutes; the tissue sections were dehydrated in a gradient manner, and after dehydration, the sections were sealed with sealing agents, and the sections were observed under a microscope and photographed, as shown in the attached Fig.33 shown.

[0818] The results showed that after antibody staining comparison, the brown (dark) area was antibody-positive, and Salmonella containing a hypoxia-specific gene expression box was found to be aggregated in the hypoxic area of ​​the tumor.

[0819] (IV) Evaluation of cell invasion of strains containing hypoxia-specific gene expression cassettes

[0820] 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.

[0821] 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).

[0822] 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 hypoxia-specific gene expression cassette on cancer cell invasion

[0823] (1) The strain modified with the hypoxia-specific gene expression cassette was transformed into the plasmid pSWT006 by electroporation to express the EGFP protein, which facilitated the observation of cell invasion.

[0824] (2) The strain carrying pSWT006 modified with the hypoxia-specific gene expression cassette was streaked on an LB plate supplemented with D-alanine and statically cultured in a 37°C constant temperature incubator overnight.

[0825] (3) A single clone of the modified strain carrying pSWT006 was selected and inoculated into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and placed in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0826] (4) Take 1OD of the culture and wash three times with PBS.

[0827] (5) Add 2.5×10 6 The corresponding cancer cells were cultured overnight in complete culture medium (DMEM basal culture medium + 10% fetal bovine serum + double antibody) in a 5% CO2 incubator at 37°C.

[0828] After counting the cells in two wells, strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038 and SWT1063 carrying pSWT006 modified with hypoxia-specific gene expression cassette were added according to the number of cells, with an infection ratio of 200:1, and placed in an anaerobic tank with an oxygen concentration below 0.8%, and co-cultured for 2 hours to allow bacteria to invade the cells. After that, the cells were carefully washed with an equal volume of PBS, and the supernatant was aspirated, and DMEM medium containing 100 μg / mL gentamicin was added. After continuing to culture in an anaerobic tank with an oxygen concentration below 0.8% for 24 hours, photos were taken under a fluorescence microscope. The invasion effects of strains modified with hypoxia-specific gene expression cassettes on different cancer cell lines are shown in the attached figure. Figure 34-63 shown.

[0829] 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 the invasion of Salmonella into cancer cells is universal.

[0830] 2. The killing effect of strains containing hypoxia-specific gene expression cassettes on cancer cells under hypoxic conditions

[0831] 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.

[0832] (1) The strain SWT005 was streaked on an LB plate supplemented with DAP; the modified strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038 and SWT1063 were streaked on an LB plate supplemented with D-alanine and cultured in a 37°C constant temperature incubator overnight.

[0833] (2) Pick a single clone of 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.

[0834] (3) Measure the absorbance of the strain at 600 nm, take the culture with an OD value of 1, wash it twice with PBS, wash it once with the corresponding cell culture medium, and then resuspend it in the cell culture medium (infection ratio is 200:1).

[0835] (4) After 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 washed three times with PBS containing gentamicin, and the medium was changed and cultured in an anaerobic tank at an oxygen concentration below 0.8% for 24 hours.

[0836] (5) Mix the CCK8 reagent with the above cell suspension and incubate for 1 hour.

[0837] (6) The absorbance was measured using an ELISA reader (detection wavelength: 450 nm) and the percentage of cytotoxicity was calculated.

[0838] Compared with the bacterium SWT005, the killing effects of the 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 were shown in the attached Figure 5 Figures 64-71 shown.

[0839] The experimental results were analyzed using T test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0840] The above results show that the Salmonella strains modified with the hypoxia-specific gene expression cassette have obvious killing effects on various cancer cells, and their effects are stronger than those of the strain SWT005 (YB1-like), indicating that the Salmonella modified with the hypoxia-specific gene expression cassette has universal killing effects on cancer cells.

[0841] 3. Targeting evaluation of strains containing hypoxia-specific gene expression cassettes in mouse tumor models

[0842] In order to explore the tumor targeting ability of the strain containing the hypoxia-specific gene expression cassette in the mouse tumor model, the EMT6 tumor mouse model was established.

[0843] BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 EMT6 cancer cells were used to establish a mouse breast subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 500mm 3 When about 30 seconds have passed, carry out the experiment.

[0844] 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 latter 16 groups were injected with 1×10 7 CFU count of bacteria. The mice were killed and dissected on the first day (D1), the fifth day (D5), and the eleventh day (D11) after administration, and the number distribution of the above strains in the normal tissues and organs of tumor-bearing mice, such as heart, liver, spleen, lung, kidney, and tumor, was detected. Results Compared with the YB1-like strain, the targeting evaluation results of each Salmonella strain containing the hypoxia-specific gene expression cassette in the mouse tumor model are shown in the attached figure. Figures 72-76 shown.

[0845] The above results show that the distribution of Salmonella strains containing hypoxia-specific gene expression boxes 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 has been completely eliminated), but they can accumulate rapidly in tumors, which is significantly higher than YB1-like strains.

[0846] The experimental results were analyzed using T test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0847] 4. Evaluation of tumor suppressive properties of strains containing hypoxia-specific gene expression cassettes in mouse tumor models

[0848] In order 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 by 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.

[0849] Establishment of EMT6 tumor mouse model: BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 EMT6 cancer cells were used to establish a mouse breast subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 500mm 3 When about 30 seconds have passed, carry out the experiment.

[0850] (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.

[0851] (2) A single clone of strain SWT005 was selected and inoculated into 5 ml of LB liquid culture medium supplemented with DAP. Single clones of constructed Salmonella strains SWT1001, SWT1002, SWT1004, SWT1005, SWT1009, SWT1010, SWT1012, SWT1013, SWT1021, SWT1025, SWT1033, SWT1035, SWT1036, SWT1038 and SWT1063 were selected and inoculated into 5 ml of LB liquid culture medium supplemented with D-alanine, and placed in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0852] (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.

[0853] (4) 1×10 7 CFU counts of bacteria were administered to EMT6 model mice.

[0854] (5) The day of drug 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 drug administration, and the mouse tumor volume was calculated according to the following formula.

[0855] Mouse tumor volume = length × width 2 ×0.52

[0856] Compared with the YB1-like strain SWT005, the tumor inhibitory effects of each Salmonella strain containing the hypoxia-specific gene expression cassette in the mouse tumor model are shown in the attached figure. Figures 77-80 shown.

[0857] The experimental results were analyzed using T test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0858] 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.

[0859] Example 3: Construction of a hypoxia-specific gene expression cassette with the dadX gene as a survival essential gene

[0860] Since there are two isozyme genes in the genome of Salmonella and Escherichia coli as alanine racemase, namely alr gene and dadX gene. In order to verify the effect, the essential gene for survival of the above-mentioned verified hypoxia-specific gene expression cassette is replaced by the corresponding homologous gene dadX gene from alr gene to identify the oxygen regulation effect of the hypoxia-specific gene expression cassette.

[0861] (I) Integrate the hypoxia-specific gene expression cassette into the alr gene locus on the chromosome of the SWT007 strain

[0862] 1. Construction of attenuated bacteria with dadX gene knockout (SWT007)

[0863] (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.

[0864] (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 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.

[0865] (3) Wash the cells three times with sterilized pure water.

[0866] (4) Preparation of PCR products of SWTO78 and SWTO79.

[0867] Primers SWTO78 and SWTO79 were mixed with plasmid pSWT002 (same as above, plasmid description), and a high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0868] 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.

[0869] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0870] (5) The recovered bacteria were mixed with 100 ng of PCR products of SWTO78 and SWTO79 and electroporated at a voltage of 1.8 kV.

[0871] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured in a 32°C incubator overnight until a single clone colony was grown.

[0872] (7) Positive clones were identified by colony PCR. SWTO59, SWTO5, SWTO6, and SWTO87 were used to identify the insertion of the chloramphenicol resistance gene. Fig.81 shown.

[0873] (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.

[0874] 2. Construction of a plasmid containing a hypoxia-specific gene expression cassette and the essential gene being the dadX gene of Salmonella

[0875] (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 mix and place it in a PCR amplification device.

[0876] 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.

[0877] (2) The amplified PCR product (Salmonella dadX gene (SEQ ID No. 19)) was purified and recovered by a DNA gel recovery system, as shown in the attached Figure 2 shown.

[0878] (3) The recovered product was mixed with HindIII and XhoI restriction enzyme system, incubated at 37°C for 1 hour, and the restriction enzyme product was purified and recovered by DNA gel recovery system.

[0879] (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.

[0880] (5) Transform into DH10B competent cells and spread on chloramphenicol and ampicillin plates to select single clones.

[0881] (6) The successfully constructed vectors are pOL2001, pOL2002, pOL2004, pOL2005, pOL2009, pOL2010, pOL2012, pOL2013, pOL2021, pOL2025, pOL2033, pOL2035, pOL2036, pOL2038 and pOL2063, as shown in Table 4.

[0882] Table 4 Combination table of forward hypoxia promoter and reverse hyperoxia promoter

[0883]

[0884] The essential gene for survival in Table 4 is the dadX gene of Salmonella.

[0885] 3. Construction of defective attenuated bacteria containing a hypoxia-specific gene expression cassette and the essential gene for survival being the dadX gene of Salmonella

[0886] (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 placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0887] (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 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.

[0888] (3) Wash the cells three times with sterilized pure water.

[0889] (4) Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette. Primers SWTO85 and SWTO86 are mixed with a hypoxia-specific gene expression cassette plasmid (as shown in Table 4) and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0890] 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.

[0891] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0892] (5) The recovered bacteria were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV.

[0893] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine and cultured in a 32°C incubator overnight until a single clone colony was grown.

[0894] (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.

[0895] (8) Use the corresponding primers to identify the clones by colony PCR. The identification results are shown in the attached Fig.82 The corresponding relationship between strains and identification primers is shown in Table 5.

[0896] Table 5 Correspondence between strains and identification primers

[0897]

[0898]

[0899] (II) Oxygen adaptability test of a library of Salmonella strains containing a hypoxia-specific gene expression cassette and the essential survival gene of Salmonella dadX (dadX gene knockout, expression cassette integrated into the Salmonella alr gene)

[0900] (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.

[0901] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0902] (3) Calculate the volume of bacterial solution per 1 OD of bacteria according to the following formula and add deionized water to 1 ml.

[0903]

[0904] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, marked as "1" and repeat three times, marked as "a", "b", and "c".

[0905] (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”.

[0906] (6) Repeat this 10-fold serial dilution until the number marked “6” is reached.

[0907] (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).

[0908] The results of the oxygen adaptability verification test of the strain containing the hypoxia specific expression cassette are shown in the attached Figures 83-86 shown.

[0909] 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.

[0910] Example 4: Study on the replacement of source bacteria for constructing a positive hypoxia promoter

[0911] In order to supplement and expand the same positive hypoxia promoter from bacteria of different origins, the present invention designed this embodiment.

[0912] (I) Positive hypoxia promoter yhbU

[0913] In the present invention, the positive hypoxia promoter used by strains SWT1001, SWT1002, SWT1004 and SWT1005 is yhbU of Salmonella. The positive hypoxia promoter used by strains SWT1065, SWT1066, SWT1068 and SWT1069 is yhbU of Escherichia coli.

[0914] For Gram-negative facultative anaerobes, the promoter yhbU contains a conserved FNR binding site, and the FNR binding site conforms to the sequence "CTGCCNNNNATCAA", where N represents any base of A, T, C, or G. The promoter yhbU conforms to the positive hypoxic promoter "TTGATNNNNATCAA", where any base in the TTGAT and ATCAA sequences in the conserved binding site can be replaced, but the total number does not exceed 3, and the 3 consecutive adjacent bases cannot be replaced.

[0915] The present invention verifies the yhbU promoters of Salmonella and Escherichia coli, and the results show that the yhbU promoters that conform to the 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.

[0916] 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 Simhuisella (SEQID No.56).

[0917] By analyzing the sequences of the yhbU promoters from the above-mentioned different sources, it was found that although the sequences of the promoters were quite different, their FNR binding sites were exactly the same. CTGCCTTAAATCAA .

[0918] Validation of oxygen adaptability of strains containing the hypoxia-specific gene expression cassette of Escherichia coli yhbU

[0919] (1) Single clones of SWT1065, SWT1066, SWT1068 and SWT1069 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.

[0920] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0921] (3) Calculate the volume of bacterial solution per 1 OD of bacteria according to the following formula and add deionized water to 1 ml.

[0922]

[0923] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, marked as "1" and repeat three times, marked as "a", "b", and "c".

[0924] (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”.

[0925] (6) Repeat this 10-fold serial dilution until the number marked “6” is reached.

[0926] (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).

[0927] The oxygen adaptability test results of Salmonella strains SWT1065, SWT1066, SWT1068 and SWT1069 are shown in the attached Fig.87 shown.

[0928] The results showed that replacing the positive hypoxia promoter from Salmonella source to Escherichia coli source can achieve similar oxygen regulation effects.

[0929] (II) Positive hypoxia promoter ynfK

[0930] The positive hypoxia promoter used by strains SWT1009, SWT1010, SWT1012 and SWT1013 is ynfK of Escherichia coli.

[0931] The positive hypoxia promoter used by strains SWT1041, SWT1042, SWT1044 and SWT1045 is ynfK of Salmonella.

[0932] For Gram-negative facultative anaerobes, the promoter ynfK contains a conserved FNR binding site, and the FNR binding site conforms to the sequence "TTGCGNNNNCTCAA", where N represents any base of A, T, C, G. The promoter ynfK conforms to the positive hypoxic promoter "TTGATNNNNATCAA", where any base in the TTGAT and ATCAA sequences in the conserved binding site can be replaced, but the total number does not exceed 3, and the 3 consecutive adjacent bases cannot be replaced.

[0933] 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.

[0934] 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).

[0935] Validation of oxygen adaptability of strains containing the hypoxia-specific gene expression cassette of the Salmonella ynfK promoter

[0936] (1) Single clones of strains SWT1041, SWT1042, SWT1044 and SWT1045 were selected and inoculated into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and placed in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0937] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0938] (3) Calculate the volume of bacterial solution per 1 OD of bacteria according to the following formula and add deionized water to 1 ml.

[0939]

[0940] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, marked as "1" and repeat three times, marked as "a", "b", and "c".

[0941] (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”.

[0942] (6) Repeat this 10-fold serial dilution until the number marked “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 oxygen adaptability test results of Salmonella strains SWT1041, SWT1042, SWT1044 and SWT1045 are shown in the attached Fig.88 The results showed that replacing the positive hypoxia promoter from Escherichia coli to Salmonella can achieve similar oxygen regulation effects.

[0945] The above results show that, for example, the two promoters yhbU and ynfK have very conserved FNR binding sites in different facultative anaerobes. By comparing the two promoters in Escherichia coli and Salmonella placed in the hypoxia-specific gene expression cassette, it was found that both have consistent oxygen regulation functions. From this, it can be judged that in different facultative anaerobes, the promoters of the same gene with conserved FNR binding site fragments can be used to construct the hypoxia-specific gene expression cassette of the present invention, and can have the same effect.

[0946] Example 5: Study on the replacement of the source bacteria for constructing the reverse hyperoxic promoter

[0947] In order to supplement and expand the same reverse hyperoxic promoter from bacteria of different origins, the present invention designed this embodiment.

[0948] (I) Reverse high oxygen promoter ydcI

[0949] In the present invention, the reverse hyperoxic promoter used by strains SWT1005, SWT1013 and SWT1021 is the ydcI promoter of Salmonella.

[0950] The reverse hyperoxic promoter used by strains SWT1008, SWT1016 and SWT1024 was the ydcI promoter of Escherichia coli.

[0951] For Gram-negative facultative anaerobes, the promoter ydcI contains a conserved FNR binding site, and the FNR binding site conforms to the sequence "CTGCCNNNNATCAA", where N represents any base of A, T, C, and G. The promoter ydcI conforms to the reverse hyperoxic promoter "TTGATNNNNATCAA", where any base in the TTGAT and ATCAA sequences in the conserved binding site can be replaced, but the total number does not exceed 3, and the 3 consecutive adjacent bases cannot be replaced; and the ArcA binding site of the reverse hyperoxic promoter conforms to the GTTAATTA pattern, where any base can be replaced, but the total number of replacements does not exceed 2.

[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 hypoxia-specific gene expression cassette containing 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 placed 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 1 OD of bacteria according to the following formula and add deionized water to 1 ml.

[0958]

[0959] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, marked as "1" and repeat three times, marked as "a", "b", and "c".

[0960] (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”.

[0961] (6) Repeat this 10-fold serial dilution until the number marked “6” is reached.

[0962] (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).

[0963] The oxygen adaptability test results of Salmonella strains SWT1008, SWT1016 and SWT1024 are shown in the attached Fig.89 shown.

[0964] The results showed that replacing the forward high oxygen promoter from Salmonella source to Escherichia coli source can achieve similar oxygen regulation effects.

[0965] (ii) The reverse hyperoxia promoter mqo containing two ArcA binding sites

[0966] In the present invention, the reverse hyperoxic promoter used by strains SWT1004, SWT1012 and SWT1036 is the argT promoter of Salmonella (SEQ ID No. 12).

[0967] The reverse high oxygen promoter used by strains SWT1073, SWT1074 and SWT1075 is the mqo promoter of Escherichia coli (SEQ ID No. 62).

[0968] Both the argT promoter (SEQ ID No. 12) and the mqo promoter (SEQ ID No. 62) contain conserved double ArcA binding sites, and also conform to the ArcA binding sites of the reverse hyperoxia promoter, conforming to the pattern of GTTAATTA, wherein any base can be replaced, but the total number of replacements does not exceed 2. The present invention verifies the argT promoter (SEQ ID No. 12) and the mqo promoter (SEQ ID No. 62), and the results prove that the promoters conforming to the conserved double ArcA binding sites all have corresponding oxygen regulation functions and can be used as the reverse hyperoxia promoter of the hypoxia-specific gene expression cassette of the present invention.

[0969] Therefore, the inverted hyperoxic promoter used in the hypoxia-specific gene expression cassette designed in the present invention may be derived from the argT promoter (SEQ ID No. 12) and the mqo promoter (SEQ ID No. 62) containing double ArcA binding sites.

[0970] Validation of oxygen adaptability of hypoxia-specific gene expression cassettes containing the mqo promoter

[0971] (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.

[0972] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0973] (3) Calculate the volume of bacterial solution per 1 OD of bacteria according to the following formula and add deionized water to 1 ml.

[0974]

[0975] (4) Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, marked as "1" and repeat three times, marked as "a", "b", and "c".

[0976] (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”.

[0977] (6) Repeat this 10-fold serial dilution until the number marked “6” is reached.

[0978] (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).

[0979] The oxygen adaptability test results of Salmonella strains SWT1073, SWT1074 and SWT1075 are shown in the attached Fig.90 shown.

[0980] The results showed that replacing the forward high oxygen promoter from Salmonella source to Escherichia coli source can achieve similar oxygen regulation effects.

[0981] 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.

[0982] Table 6 Specific sequences of the forward hypoxia promoter, survival essential gene and reverse hyperoxia promoter of the present invention

[0983]

[0984]

[0985]

[0986]

[0987]

[0988]

[0989]

[0990]

[0991]

[0992]

[0993]

[0994]

[0995] The specific sequences of all primers used in the present invention are shown in Table 7.

[0996] Table 7 Specific sequences of all primers used in the present invention

[0997]

[0998]

[0999]

[1000]

[1001] The tool plasmids of the present invention are shown in Table 8.

[1002] Table 8 Tool plasmids used in the present invention

[1003]

[1004] The present application has been described above in conjunction with preferred embodiments, but these embodiments are only exemplary and serve only as an illustration. On this basis, various replacements and improvements may 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 inverse hyperoxic promoter is selected from the promoter region sequence of cyoA, ydcI, phoH, argT, mqo or lldP. 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.

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