Constitutive expression vector containing dual bacteriophage-derived promoters, bacterium containing same, and uses thereof

Abstract

A constitutive expression vector containing dual bacteriophage-derived promoters, a gram-negative bacterium containing the expression vector, and uses thereof in preparation of tumor drugs and treatment of tumors. By selecting bacteriophage-derived promoters, a specific bacteriophage RNA polymerase gene, a membrane-disrupting protein gene, and a therapeutic protein gene, the expression vector is divided into a protein expression control module, a drug delivery module, and a drug-mediated killing module. By organically combining the three modules, a three-in-one system is formed, and a system and method for stable and efficient drug protein expression, drug delivery, and drug-mediated killing are realized.

Description

A constitutive expression vector containing a double phage-derived promoter, bacteria containing the same, and applications thereof

[0001] This application claims priority to a prior application, patent application number 202311480978.8, filed with the State Intellectual Property Office of China on November 8, 2023, entitled “A constitutive expression vector containing a dual phage-derived promoter, bacteria containing the same, and applications thereof.” The entire text of that prior application is incorporated herein by reference. Technical Field

[0002] The present invention relates to a constitutive expression vector containing a double phage-derived promoter, bacteria containing the same and applications thereof, and belongs to the fields of genetic engineering and biotherapy. Background Art

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

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

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

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

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

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

[0009] Regarding improvements in targeting tumors with recombinant bacteria, existing literature reports indicate that these bacteria can also carry different types of drugs for targeted drug delivery to tumors. However, these efforts have been less than ideal. This may be due to a lack of comprehensive consideration of the efficiency of bacterial drug synthesis and delivery within tumors. The following three points require careful consideration:

[0010] First, the intensity and stability of target drug expression.

[0011] The main factor considered in previous bacterial in vitro expression technologies was to achieve high expression of the target protein under stable culture conditions, such as high-density culture in a nutrient-rich culture medium. However, if the bacteria are in an in vivo tumor environment, the bacteria themselves will face the pressure of a complex tumor microenvironment (including extreme environments such as hypoxia, low pH, high lactic acid, and immune cells targeting bacteria), and their growth and the synthesis of the target protein will be affected. In addition, the promoter of the bacteria itself will be affected by external factors, such as chemicals, environment, temperature or other regulatory factors. Therefore, directly using the natural promoter of bacteria or adopting traditional bacterial high expression strategies may affect the expression of the target drug protein. In addition, due to the limited capacity of bacterial chromosomes, most genes exist in the form of single genes, and the strength of natural bacterial promoters is not high, so the expression level of drug proteins will also be affected, thereby affecting the effect of tumor inhibition.

[0012] Second, the choice of medicine is very important.

[0013] The environment inside a tumor is not completely closed. Therefore, if anti-tumor drugs are released directly into the tumor, some of the drugs will diffuse through blood vessels into other organs and tissues, causing unnecessary side effects. Therefore, it is particularly important to select a drug that can kill tumor cells only within the tumor cell.

[0014] Third, the method of drug delivery is very important.

[0015] To deliver drugs into tumor cells, bacteria must first invade the tumor cells and then release the drugs inside them. Since bacteria invade cells by inducing endocytosis, forming phagocytic vesicles, the drugs synthesized by bacteria need to pass through these vesicles to reach the tumor cell cytoplasm and take effect. Furthermore, the release efficiency of natural bacteria is very low. Therefore, a highly efficient drug release mode that penetrates the vesicles is needed to increase the drug's effectiveness.

[0016] In summary, there is an urgent need for a drug protein system and method that can enable bacteria to stably, efficiently express, and efficiently release drug proteins in tumors in vivo.

[0017] Summary of the Invention

[0018] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terminology and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the relevant fields and are standard procedures.

[0019] In response to the above-mentioned defects in the prior art, the present invention establishes a three-in-one system for stably and efficiently performing drug protein expression, drug delivery, and drug killing in bacteria.

[0020] To this end, the present invention provides a constitutive plasmid expression vector, which contains two phage-derived promoters that control the expression of drug protein genes and membrane-breaking protein genes respectively, and integrates a specific phage RNA polymerase gene controlled by the constitutively expressed promoter on the host bacterial chromosome.

[0021] The constitutive plasmid expression vector of the present invention comprises three modules, namely: (1) a protein expression control module; (2) a drug delivery module; and (3) a drug killing module.

[0022] By organically combining the above three modules and coordinating with the characteristics of bacteria, the constitutive plasmid expression vector of the present invention realizes a three-in-one system for stable and efficient drug protein expression, drug delivery and drug killing in bacteria.

[0023] (1) Protein expression control module

[0024] In order to achieve stable and efficient gene expression in the tumor microenvironment, a high-strength promoter expression system that is not affected by the environment is required.

[0025] The present invention preferably adopts constitutive artificial promoter lacUV5 to control the expression of phage-derived T7 RNA polymerase gene, and this expression cassette is integrated into the chromosome of bacteria to achieve stable inheritance. Due to the T7 RNA polymerase specific recognition T7 promoter, the gene controlled by the T7 promoter can be achieved to be stable and highly expressed. Therefore, the present invention selects that the drug delivery module gene and the drug killing module gene are respectively placed behind the T7 promoter, and are placed in a plasmid expression vector for expression regulation.

[0026] (2) Drug delivery module

[0027] When bacteria invade tumor cells, they form phagocytic vesicles. The presence of these vesicles limits the release of the bacteria and the drugs they synthesize. To address this issue, the present invention utilizes listeriolysin O (LLO) as a membrane-permeabilizing protein for drug delivery.

[0028] (3) Drug killing module

[0029] In order to achieve precise drug delivery that only exerts a killing effect within tumor cells, the present invention uses the A fragment of the bacterial AB type exotoxin protein to achieve a precise intracellular killing effect. Because the characteristic of the bacterial AB type exotoxin protein is that the exotoxin protein can be divided into two independent functional regions, the characteristic of the B fragment is that it can specifically bind to the extracellular receptors of the target cell, induce changes in the cell membrane, and release the A fragment into the cytoplasm. The A fragment can only exert a cytotoxic effect in the cytoplasm, and its mechanism of action is related to bacterial exotoxin genes from different sources. Therefore, if the expression vector only uses the A fragment, when the excess A fragment is released into the human circulatory system, no toxic side effects will be produced.

[0030] In the process of tumor treatment using the constitutive plasmid expression vector of the present invention, bacteria enter the tumor tissue through the circulatory system and colonize therein. Inside the bacteria, T7 RNA polymerase is stably expressed, promoting the stable and high expression of the killer drug exotoxin A fragment and the drug delivery module membrane-breaking protein LLO located on the plasmid. When the bacteria approach cancer cells in the tumor tissue, the bacteria use their own invasion system to induce the cancer cells to undergo cell membrane endocytosis, forming phagocytic vesicles containing bacteria. Thereafter, the phagocytic vesicles will fuse with vacuoles such as lysosomes in the cell, thereby lowering the pH inside the phagocytic vesicles. At this time, the membrane-breaking protein LLO expressed by the bacteria is activated, causing the killer drug exotoxin A fragment to be released into the cytoplasm, ultimately leading to the death of cancer cells. This process is repeated as the bacteria grow and replicate inside the tumor, causing the rapid digestion of tumor cells.

[0031] It can be seen that the present invention achieves stable and efficient drug protein expression, drug delivery and drug killing in bacteria based on the perfect combination of the above three-in-one system.

[0032] Therefore, in the present invention, a promoter containing two phage sources is used to control the expression of the drug protein exotoxin A fragment and the membrane-permeabilizing protein gene respectively, and the phage-derived promoter binds to the RNA polymerase encoded by the specific phage RNA polymerase gene on the host bacterial chromosome.

[0033] In a preferred embodiment of the present invention, the dual phage-derived promoter is selected from the group consisting of T7 (SEQ ID No. 1), T3 (SEQ ID No. 2) and SP6 promoter (SEQ ID No. 3).

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

[0035] In addition, according to the different directions of the dual phage-derived promoters, the preferred expression vector of the present invention has the dual phage-derived promoters in opposite directions, which is an opposing promoter expression vector, as shown in FIG18 .

[0036] In addition, to prevent the loss of the plasmid expression vector, an essential gene of Salmonella needs to be implanted during the construction of the vector, and the gene needs to be removed from the chromosome of the host bacteria at the same time, thereby forming a balanced lethal control mechanism. That is, if the Salmonella loses the plasmid vector it carries, the bacteria will die quickly due to the lack of essential genes, and the surviving bacteria will carry the plasmid expression vector, thus ensuring that the entire system can stably and efficiently express protein drugs.

[0037] The Salmonella asd gene (SEQ ID No. 11) and its protein sequence (SEQ ID No. 12) encode aspartate β-semialdehyde dehydrogenase, an enzyme required for the synthesis of diaminopimelate (DAP), a key component of the cell wall of Gram-negative bacteria. Deleting the asd gene in Salmonella results in lytic death of the bacteria. However, supplementing the culture medium with DAP or by transfecting Salmonella with a vector containing the asd gene maintains normal growth.

[0038] Therefore, in a preferred embodiment of the present invention, the expression vector contains the essential gene asd of Salmonella, and the asd gene on the chromosome of the host bacteria is deleted.

[0039] In order to achieve efficient expression of the expression vector, the expression vector of the present invention preferably contains a replicon necessary for plasmid replication.

[0040] Meanwhile, the expression vector of the present invention does not contain a resistance gene.

[0041] In a preferred embodiment of the present invention, the replicon is selected from the group consisting of pUC, p15A, ColE1 and R6K.

[0042] In a more preferred embodiment of the present invention, the replicon is pUC (SEQ ID No. 36).

[0043] In order to carry out drug killing stably and efficiently, the present invention selects drug proteins with high efficiency and specific killing power, which can kill tumor cells while preventing accidental damage to other organs after drug protein leakage.

[0044] Therefore, in a preferred embodiment of the present invention, the drug protein gene is the A fragment of the AB type exotoxin drug gene of bacterial origin.

[0045] In a preferred embodiment of the present invention, the pharmaceutical protein gene used in the present invention is the diphtheria toxin A fragment (DTA) (SEQ ID No. 15) and protein sequence (SEQ ID No. 16) after Salmonella codon optimization.

[0046] Diphtheria toxin (DT) is a protein toxin composed of two functionally independent subunits (A and B) linked by a disulfide bond.

[0047] The A subunit (DTA) has enzymatic activity and can inactivate elongation factor-2 (EF-2) by catalyzing the glycosylation of ADP, thereby blocking protein synthesis in eukaryotic cells. DTA is the main source of diphtheria toxin cytotoxicity.

[0048] The B subunit (DTB) is non-cytotoxic and binds to receptors on the surface of eukaryotic cells, mediating the entry of the A subunit into cells. Due to the independent functions of diphtheria toxin DTA and DTB, when DTA is independently expressed in bacteria but does not enter eukaryotic cells, it does not harm bacteria or eukaryotic cells. Toxicity to eukaryotic cells only occurs when the DTA protein is released and enters the cells.

[0049] In addition, in other preferred embodiments of the present invention, the drug protein gene can also be selected from other AB-type bacterial exotoxin genes with similar functions to A subunits and B subunits. For example, the cholera toxin A subunit fragment gene (SEQ ID No. 17) and its protein sequence (SEQ ID No. 18), the Shiga toxin gene Stx1 A fragment (SEQ ID No. 19) and its protein sequence (SEQ ID No. 20), the Shiga toxin gene Stx2 A fragment (SEQ ID No. 21) and its protein sequence (SEQ ID No. 22), the pertussis toxin A subunit fragment gene (SEQ ID No. 23) and its protein sequence (SEQ ID No. 24), and the Pseudomonas exotoxin A subunit fragment gene (SEQ ID No. 25) and its protein sequence (SEQ ID No. 26).

[0050] Cholera toxin (CT) originates from Vibrio cholerae and is composed of two subunits, A and B. Subunit A is an ADP-ribosyltransferase that disrupts G protein signaling, leading to cell dehydration and death. Cholera toxin subunit B (CTB) binds to the pentasaccharide chain of ganglioside GM1, attaching to the cell surface. It is then taken up by axon terminals and transported back to the cell body.

[0051] Shiga toxin genes are a family of related toxins with two major classes: Stx1 and Stx2. The toxins consist of two subunits, A and B, which are members of the AB toxin family. The B subunit is a pentamer that binds to specific glycolipids on host cells, particularly triacylceramide (Gb3). Binding of the B subunit to Gb3 induces narrow tubular membrane invaginations, driving the formation of endometrial tubules for bacterial uptake into the cell. Once inside the cell, the A subunit cleaves specific adenine nucleobases from the 28S RNA of the 60S ribosomal subunit, halting protein synthesis.

[0052] Pertussis toxin (PT) is a protein-based AB-type exotoxin produced by Bordetella pertussis that causes whooping cough. The A subunit of PT is enzymatically active and is translocated into host cells following a conformational change in the membrane-bound transporter B subunit. Pertussis toxin is an exotoxin composed of six subunits (designated S1 to S5, with each complex containing two copies of S4). The subunits are arranged in an AB structure: the enzymatically active A component is formed by the S1 subunit, while the receptor-binding B component is composed of subunits S2-S5. PT is released from B. pertussis in an inactive form. After binding to cell membrane receptors, PT is taken up by endosomes and then retrogradely transported to the trans-Golgi network and endoplasmic reticulum. During transport, the A subunit is activated by glutathione and ATP, catalyzing the ADP-ribosylation of the heterotrimeric G protein. Consequently, it is unable to inhibit adenylate cyclase activity, leading to increased intracellular cAMP concentrations and cell death.

[0053] Pseudomonas exotoxin is an AB type exotoxin produced by Pseudomonas aeruginosa. + The toxin transfers adenosine diphosphate ribose (ADP-ribose) on the target protein to a process called ADP-ribosylation modification. The pathogenic mechanism is to inhibit eukaryotic elongation factor 2 (EF2) by ADP-ribosylating the diphtheria amide residue of EF2, thereby stopping the elongation of the polypeptide (the mechanism of the toxin is similar to that of diphtheria toxin).

[0054] To achieve stable and efficient drug delivery, the present invention designs a precise drug delivery system. Its goal is to precisely release the drug protein synthesized by bacteria into tumor cells. This process is further divided into two steps: bacteria invading host cancer cells and releasing the synthesized drug protein.

[0055] In a preferred embodiment of the present invention, the natural ability of Salmonella to invade host animal cells is utilized to achieve bacterial invasion of host cancer cells.

[0056] Salmonella enterica is capable of invading and replicating within host cells, including epithelial cells and macrophages. This ability is embodied in the pathogenicity island 1 of Salmonella. Through the type III secretion system 1 (T3SS1), Salmonella secrete T3SS effector proteins, which induce extensive actin rearrangements within the host cell, leading to membrane ruffling and the formation of phagocytic vesicles containing the Salmonella. The type III secretion system 2 (T3SS2), encoded by the Salmonella pathogenicity island 2 (SPI-2) gene, then promotes the survival and proliferation of Salmonella within the phagocytic vesicles.

[0057] In a preferred embodiment of the present invention, the purpose of releasing the synthetic drug protein is achieved by using the listeriolysin-O (LLO) gene (SEQ ID No. 13) and protein sequence (SEQ ID No. 14) synthesized by Salmonella and optimized by Salmonella codons.

[0058] LLO is encoded by the hlyA gene of Listeria monocytogenes (LM) and can bind to the cholesterol in the host cell membrane to form a pore-like structure with a diameter of 35 nm.

[0059] LLO has a pH-sensitive acidic domain. The optimal pH for LLO precursor protein maturation and the onset of membrane perforation activity is approximately 5.0-5.5, while it is inactivated at neutral pH.

[0060] When Salmonella invades and forms phagocytic vesicles, they fuse with intracellular vacuoles such as lysosomes, significantly lowering the pH. Under these conditions, LLO activity is activated, exerting a perforating effect, allowing Salmonella to release the synthesized drug protein.

[0061] Therefore, in a preferred embodiment of the present invention, the membrane permeabilin gene is the listeriolysin hlyA gene.

[0062] In a preferred embodiment of the present invention, in the dual phage-derived promoter, one is sequentially connected to the ribosome binding site (RBS) (SEQ ID No. 35) and the membrane permeabilin gene, and the other is sequentially connected to the ribosome binding site (RBS) (SEQ ID No. 35) and the drug protein gene.

[0063] Furthermore, in a preferred embodiment of the present invention, the host bacteria of the expression vector are Gram-negative bacteria.

[0064] Another aspect of the present invention provides a method for constructing the plasmid expression vector of the present invention, comprising the following steps:

[0065] 1. Sequentially connect the phage-derived promoter, ribosome binding site and membrane-permeabilizing protein gene described in the present invention to construct a membrane-permeabilizing protein gene expression unit;

[0066] 2. Sequentially connect the phage-derived promoter, ribosome binding site and drug protein gene described in the present invention to construct a drug protein gene expression unit;

[0067] 3. Connect the plasmid replicon, asd gene, and the above two gene expression units together to form a complete plasmid expression vector.

[0068] Another aspect of the present invention provides a modified Gram-negative bacterium comprising the expression vector of the present invention.

[0069] In a preferred embodiment of the present invention, the specific phage RNA polymerase gene is a gene corresponding to a promoter derived from a biphage.

[0070] In a more preferred embodiment of the present invention, the specific phage RNA polymerase gene is selected from the T7 RNA polymerase gene (SEQ ID No.4) and T7 RNA polymerase protein sequence (SEQ ID No.5) after Salmonella codon optimization, the T3 RNA polymerase gene (SEQ ID No.6) and T3 RNA polymerase protein sequence (SEQ ID No.7) and the SP6 RNA polymerase gene (SEQ ID No.8) and SP6 RNA polymerase protein sequence (SEQ ID No.9).

[0071] In a further preferred embodiment of the present invention, the specific bacteriophage RNA polymerase gene is a T7 RNA polymerase gene.

[0072] In order to stably and efficiently express the drug protein, the present invention places the drug protein gene on a multi-copy plasmid expression vector and controls it through a T7 promoter to achieve maximum expression of the drug protein gene.

[0073] In order to achieve higher intensity expression, in a preferred embodiment of the present invention, a T7 phage RNA polymerase expression system is used, which includes T7 RNA polymerase and T7 promoter to control the expression of the target drug protein.

[0074] The T7 RNA polymerase system, derived from the expression system of the Escherichia coli T7 bacteriophage, is widely used in E. coli gene expression due to its robust protein expression capabilities. T7 RNA polymerase is characterized by its ability to specifically recognize the T7 promoter and is unaffected by environmental factors. Furthermore, T7 RNA polymerase's RNA synthesis rate is five times that of E. coli RNA polymerase, enabling high-intensity expression of target proteins.

[0075] In a preferred embodiment of the present invention, the T7 polymerase and T7 promoter can be replaced with T3 polymerase and T3 promoter. Similar to the T7 RNA polymerase system, the T3 RNA polymerase system is an RNA polymerase system derived from T3 bacteriophage that highly specifically recognizes the T3 promoter sequence. Alternatively, the T7 polymerase and T7 promoter can be replaced with SP6 polymerase and SP6 promoter. Similar to the T7 RNA polymerase system, SP6 polymerase is also an RNA polymerase system derived from SP6 bacteriophage that highly specifically recognizes the SP6 promoter sequence. T3 RNA polymerase or SP6 RNA polymerase and its corresponding promoter can functionally replace T7 RNA polymerase to specifically control the expression of downstream target genes.

[0076] In order to achieve stable expression without being affected by bacterial internal metabolism and the tumor microenvironment, in a preferred embodiment of the present invention, the expression of the specific phage RNA polymerase gene is regulated by a constitutive expression promoter.

[0077] In a more preferred embodiment of the present invention, a constitutively expressed lacUV5 promoter (SEQ ID No. 10) is further used to control the expression of a specific bacteriophage RNA polymerase gene.

[0078] The lacUV5 promoter is very similar to the classic lac promoter. Compared to the lac promoter, it contains only a two-base-pair mutation in the -10 region. The lacUV5 promoter does not require an additional activator and can drive high levels of gene expression. Although it does not require an activator, expression from the lacUV5 promoter is regulated by the LacI repressor in E. coli and is inducible by IPTG. IPTG is an effective inducer when used in concentrations ranging from 100 μM to 1.5 mM.

[0079] Since Salmonella has lost the lacI gene and the entire lac operon during evolution, the lacUV5 promoter is an excellent constitutive expression system in Salmonella.

[0080] Therefore, in a more preferred embodiment of the present invention, a T7 RNA polymerase expression cassette controlled by a lacUV5 promoter is further transferred into the chromosome of Salmonella, thereby achieving stable constitutive expression.

[0081] In a preferred embodiment of the present invention, the Gram-negative bacteria further comprises a hypoxia-specific gene expression cassette comprising:

[0082] 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;

[0083] b) genes essential for survival; and

[0084] c) an inverted hyperoxic promoter, which is a promoter comprising FNR and ArcA binding sites, and the inverted hyperoxic promoter can function under the oxygen content conditions of normal organs;

[0085] The essential gene for survival is a gene encoding alanine racemase.

[0086] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0087] In the present invention, "polynucleotide" refers to a macromolecule composed of multiple nucleotides linked by phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present invention can be codon-optimized for different host cells (e.g., Escherichia coli) to improve polypeptide expression. Methods for codon optimization are known in the art.

[0088] "Sequence identity" between two polypeptide sequences or two polynucleotide sequences refers to the percentage of identical amino acids or nucleotides between the sequences. Methods for assessing the level of sequence identity between polypeptide or polynucleotide sequences are known in the art. Sequence identity can be assessed using various known sequence analysis software. For example, sequence identity can be assessed using the EMBL-EBI online alignment tool (https: / / www.ebi.ac.uk / Tools / psa / ). The sequence identity between two sequences can be assessed using the Needleman-Wunsch algorithm using default parameters.

[0089] In order for bacteria to survive only in low oxygen concentrations, three requirements need to be met:

[0090] 1) Control the strength of the upstream promoter and background leakage expression.

[0091] If the oxygen sensor is partially leaky without binding to the upstream promoter, it will be impossible to regulate oxygen, resulting in the bacteria surviving at any oxygen concentration. Alternatively, if the upstream promoter is too weak, it will be unable to initiate the expression of downstream genes, resulting in the bacteria not surviving at any oxygen concentration.

[0092] 2) Select appropriate genes essential for survival.

[0093] By selecting genes essential for survival, it is possible to ensure that the bacteria die when the genes are not expressed, and to quickly ensure bacterial survival when the genes are induced by hypoxia;

[0094] 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.

[0095] Therefore, in the present invention, a "hypoxia-specific gene expression cassette" refers to a segment of DNA that can initiate expression of an essential gene under hypoxic conditions, which contains an essential gene controlled by a hypoxia-inducible promoter and, if necessary, may further contain other regulatory elements required for the expression of the essential gene.

[0096] In the present invention, an "essential gene" refers to a gene that plays a crucial role in bacterial growth and / or survival. If the bacterium lacks this gene or its functional expression product, it will not survive, divide, and / or grow normally. Typical examples of bacteria lacking essential genes or their functional expression products are auxotrophic strains, which, in the absence of specific exogenous supplements, cannot survive, divide, and / or grow normally under in vitro culture conditions or in vivo environments. Essential genes are typically present in a single copy on the bacterial chromosome.

[0097] From this, we can see that the requirements for genes essential for survival are as follows:

[0098] (1) It is an essential gene for bacterial reproduction. If it is missing, it will lead to rapid death of bacteria;

[0099] (2) The product of this gene does not exist in the normal environment or the human body, which ensures that it will not get out of control in the human environment, and can be easily cultured and prepared by adding the corresponding expression product of this gene in a normal culture environment;

[0100] (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.

[0101] In the present invention, the essential gene for survival is a gene encoding alanine racemase.

[0102] The cell wall is an essential component of Gram-negative bacteria such as Escherichia coli and Salmonella. The core component of the cell wall is peptidoglycan. To synthesize peptidoglycan, bacteria require D-alanine, a crucial building block. Without D-alanine, bacteria cannot synthesize their cell wall and subsequently undergo cell lysis.

[0103] All amino acids exist in nature are L-type, so there are two genes in Gram-negative bacteria: the alr gene for the biosynthesis of alanine racemase and dadX, which are responsible for converting L-alanine into D-alanine to meet the needs of cell wall synthesis.

[0104] 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.

[0105] In our previous research, YB1 Salmonella used the asd gene as an essential gene for regulation. Compared to YB1 Salmonella, the present invention selected the alr gene and dadX gene as essential genes for survival. The alr gene and dadX gene are functionally homologous genes. Therefore, the gene for alanine racemase in the present invention can be the alr gene (SEQ ID No. 27) and the protein sequence of alr from Salmonella (SEQ ID No. 28), or the dadX gene (SEQ ID No. 29) and the protein sequence of dadX from Salmonella (SEQ ID No. 30); or the alr or dadX gene from other Gram-negative bacteria, or genes with equivalent functions.

[0106] In the present invention, the alanine racemase alr gene and dadX gene comprise nucleotide sequences having a sequence identity of greater than or equal to 81%, preferably greater than or equal to 82%, more preferably greater than or equal to 83%, greater than or equal to 84%, greater than or equal to 85%, greater than or equal to 86%, greater than or equal to 87%, greater than or equal to 88%, greater than or equal to 89%, greater than or equal to 90%, greater than or equal to 91%, greater than or equal to 92%, greater than or equal to 93%, greater than or equal to 94%, greater than or equal to 95%, greater than or equal to 96%, greater than or equal to 97%, greater than or equal to 98%, or most preferably greater than or equal to 99% to the above-mentioned specific sequences.

[0107] In the present invention, alr gene knockout bacteria were first constructed, and then another gene, dadX, was modified, so that the bacteria became deficient in alr and dadX genes after editing. At the same time, a forward hypoxia promoter and a reverse hyperoxia promoter were used to regulate the additional alr or dadX gene.

[0108] The positive hypoxic promoter described herein is a hypoxic promoter regulated by FNR; the reverse hyperoxic promoter described herein is an antisense promoter negatively regulated by FNR and ArcA. The fumarate and nitrate reduction gene fnr is a key gene regulating aerobic and anaerobic growth in Salmonella. This complex regulatory system has been extensively studied in Escherichia coli and Salmonella. The DNA-binding protein FNR encoded by the fnr gene senses changes in oxygen and controls the expression of various genes, achieving overall metabolic shifts. Therefore, DNA-binding sequences such as FNR and ArcA have become important mechanisms for controlling downstream gene expression.

[0109] FNR has an oxygen-sensitive [4Fe-4S] 2+ The FNR domain directly senses oxygen and regulates site-specific DNA binding. In contrast, ArcA senses signals from the aerobic respiratory chain. Therefore, Gram-negative facultative anaerobes such as Salmonella and Escherichia coli utilize two distinct mechanisms for sensing changes in oxygen concentration. FNR regulates gene expression in response to hypoxia, either through activation or repression.

[0110] To this end, the present invention uses the forward hypoxia promoters yhbU and ynfK that FNR activates downstream gene expression under hypoxia, and the reverse hyperoxia promoters ydcI and cyoA that FNR and ArcA inhibit downstream gene expression under hypoxia.

[0111] Among them, the positive hypoxia promoter used in the present invention is as follows:

[0112] (1) Salmonella yhbU promoter (yhbU-S) (SEQ ID No. 31), which contains the FNR binding site "CTGCCTTAAATCAA";

[0113] (2) Escherichia coli ynfK promoter (ynfK-E) (SEQ ID No. 32), wherein the FNR binding site is "TTGCGCTATCTCAA";

[0114] Among them, the reverse hyperoxia promoter used in the present invention is as follows:

[0115] (1) Salmonella cyoA promoter (cyoA-S) (SEQ ID No. 33), which contains the FNR binding site "TTTATTGATAATAA" and the ArcA binding site "GTTAAGTA";

[0116] (2) Salmonella ydcI promoter (ydcI-S) (SEQ ID No. 34), which contains the FNR binding site "GTTATCAAAAACAA" and the ArcA binding site "GTTAATAA";

[0117] Through analysis of the above-mentioned reverse hypoxia promoter and forward hyperoxia promoter, we found that the FNR binding sites all conform to the "TTGATNNNNATCAA" pattern, and any base in the TTGAT and ATCAA sequences in the conserved binding sites can be replaced, but the total number does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; and ArcA conforms to the "GTTAATTA" pattern of its core region, and any base in the GTTAATTA sequence in the conserved binding site can be replaced, but the total number does not exceed 2.

[0118] Therefore, in a preferred embodiment of the present invention, the FNR binding site of the forward hypoxia promoter or the reverse hyperoxia promoter conforms to the pattern of TTGATNNNNATCAA, where N is any base of A, T, C, or G, and any base in the TTGAT or ATCAA sequence in the conserved binding site can be replaced, but the total number of replacements does not exceed 3, and three consecutive adjacent bases cannot be replaced; the ArcA binding site of the reverse hyperoxia promoter conforms to the pattern of GTTAATTA, wherein any base can be replaced, but the total number of replacements does not exceed 2.

[0119] In a preferred embodiment of the present invention, the forward hypoxia promoter and / or the reverse hyperxia promoter of the hypoxia-specific gene expression cassette is a promoter derived from Gram-negative bacteria.

[0120] In a more preferred embodiment of the present invention, the forward hypoxia promoter is selected from the promoter region sequences of yhbU and ynfK; the survival essential gene is selected from alr and dadX; and the reverse hyperoxia promoter is selected from the promoter region sequences of cyoA and ydcI.

[0121] 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 Simihuiella.

[0122] 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.

[0123] In a further preferred embodiment of the present invention, the alr gene and the dadX gene are selected from the group consisting of Salmonella, Escherichia coli, Shigella, Klebsiella, Yersinia, Haemophilus and Pseudomonas.

[0124] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter cyoA.

[0125] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter yhbU, the survival essential gene alr and the reverse hyperoxia promoter ydcI.

[0126] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene dadX and the reverse hyperoxia promoter cyoA.

[0127] In a further preferred embodiment of the present invention, the expression cassette consists of the forward hypoxia promoter ynfK, the survival essential gene dadX and the reverse hyperoxia promoter ydcI.

[0128] The positive hypoxia promoter disclosed in patent CN104471057B is pepT. The pepT promoter is not completely regulated by FNR, but is a promoter that is dual-regulated by CRP-cAMP and FNR. Therefore, half of the FNR binding site in the pepT promoter is a CRP binding site, and half is a FNR binding site. The CRP-FNR binding region sequence of the pepT promoter is GTGACCTGACGCAA, of which the first half GTGA conforms to the first half of the CRP conserved binding site GTGANNNNNNTCAC, and the second half CGCAA conforms to the ATCAA portion of the FNR conserved region. The A at the tenth position is replaced by C, and the T at the eleventh position is replaced by G, which does not conform to the rules for positive hypoxia promoters in the present invention.

[0129] The FNR binding region of the reverse hyperoxia promoter sodA, disclosed in patent CN104471057B, contains only the first half of the FNR region, TTGAT, while the second half contains three consecutive substitutions of ATCAA with ATTTT, thus not containing a complete FNR binding site. The ArcA binding site is TTTAATTA, which, compared to the conserved core ArcA binding site 5'-GTTAATTA-3', has the first G replaced by a T. Therefore, the sodA promoter contains only a single ArcA binding site, and its FNR binding site is incomplete, which does not meet the requirements of the present invention for reverse hyperoxia promoters.

[0130] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is regulated by oxygen concentration.

[0131] For Gram-negative bacteria like Salmonella to survive in a hypoxic zone, an oxygen concentration of 1% is crucial, representing pathological hypoxia. This is because oxygen concentrations below 1% are a clear marker of a tumor hypoxic zone. In normal organs and tissues, there are no areas with oxygen concentrations below 1%. For example, in pancreatic cancer, cervical cancer, prostate cancer, and other tumors, the oxygen concentration in the hypoxic zone is less than 0.7%.

[0132] The hypoxia-specific gene expression cassette designed in this invention precisely regulates oxygen levels, enabling Salmonella and other Gram-negative bacteria to recognize and proliferate in hypoxic tumor areas. The goal of this invention is to enable the modified Salmonella and other Gram-negative bacteria to survive and proliferate in oxygen concentrations below 0.8%, and to achieve suicide lysis in normal oxygen concentrations.

[0133] Thus, in a more preferred embodiment of the present invention, the positive hypoxia promoter functions when the oxygen content is below 1%, but fails to function when the oxygen content is above 1%; and / or

[0134] The inverted hyperoxic promoter is functional when the oxygen content is above 1%, but is inactive when the oxygen content is below 1%.

[0135] 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

[0136] The inverted hyperoxic promoter is functional when the oxygen content is above 0.8%, but is inactive when the oxygen content is below 0.8%.

[0137] Another aspect of the present invention provides a method for controlling the expression of a pharmaceutical protein in prokaryotic cells using the expression vector of the present invention, comprising the following steps:

[0138] 1. Prepare the expression vector of the present invention;

[0139] 2. Integrate the constitutively expressed bacteriophage RNA polymerase gene expression cassette into the host bacterial chromosome;

[0140] 3. Knock out the asd gene on the host bacterial chromosome;

[0141] 4. Transform the expression vector of the present invention into host bacteria.

[0142] In a preferred embodiment of the present invention, the method further comprises integrating the hypoxia-specific gene expression cassette of the present invention into the host bacterial chromosome.

[0143] In a preferred embodiment of the present invention, the host bacterium is a Gram-negative bacterium.

[0144] In the present invention, term " gram-negative bacteria " refers to after carrying out the known partial program of gram staining, does not retain the bacterium of the initial basic dye staining agent (such as, crystal violet) as.In exemplary gram staining, first by heating, cell is fixed on slide and dyed with basic dye (such as, crystal violet), and described basic dye is absorbed by both gram-negative bacteria and gram-positive bacteria.Then, slide is processed with mordant (such as, Gram's iodine solution), and described mordant is incorporated into basic dye (such as, crystal violet) and is trapped in cell.Then, by cell acetone or ethanol washing, then with the second dye of different colors counterstaining (such as, safranin).Gram-positive organism retains initial purple dyeing, and gram-negative organism decolorizes by organic washing solvent, therefore shows counterstaining. Exemplary Gram-negative bacteria include, but are not limited to, Escherichia species, Shigella species, Salmonella species, Campylobacter species, Neisseria species, Haemophilus species, Aeromonas species, Francisella species, Yersinia species, Klebsiella species, Bordetella species, Legionella species, Corynebacterium species, Citrobacter species, Chlamydia species, Brucella species, Pseudomonas species, Helicobacter species, and Vibrio species.

[0145] 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, Simihuisella, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida , Legionella, Klebsiella pneumoniae, Vibrio parahaemolyticus, Vibrio cholerae, Yersinia pestis, Coccus catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia and Helicobacter pylori.

[0146] In the present invention, the term "live bacteria" refers to a strain that has vitality, active nutritional metabolic characteristics, and is able to perform its own biological functions. Live bacteria can include bacterial biomass produced during the metabolic process of the strain.

[0147] In a more preferred embodiment of the present invention, the Gram-negative bacteria is Salmonella.

[0148] When the expression vector of the present invention is introduced into Gram-negative bacteria (e.g., Salmonella typhimurium) as a therapeutic bacterium, the efficient expression of the drug protein gene and membrane-permeabilizing protein gene controlled by the specific phage RNA polymerase and the dual phage-derived promoter integrated into the bacterial chromosome will lead to increased bacterial toxicity. Therefore, in order to achieve safety as a therapeutic bacterium, the bacteria integrated with the expression vector of the present invention need to be modified by one or more attenuation methods. Attenuation methods include, but are not limited to, hypoxia-specific gene expression cassette regulation systems, nutritional deficiencies, stress response defects, and virulence island regulation defects.

[0149] Therefore, in a preferred embodiment of the present invention, the Gram-negative bacteria are attenuated Gram-negative bacteria.

[0150] In a more preferred embodiment of the present invention, the attenuated Gram-negative bacterium is Salmonella.

[0151] In a more preferred embodiment of the present invention, the attenuation method is selected from the group consisting of aroA gene deficiency of Salmonella and a hypoxia-specific gene expression cassette regulation system.

[0152] Another aspect of the present invention provides use of the expression vector or the Gram-negative bacteria of the present invention in the preparation of tumor drugs.

[0153] In a preferred embodiment of the present invention, the tumor is a solid tumor.

[0154] The term "solid tumor" used in the present invention refers to an abnormal mass of tissue, generally not comprising a cyst or liquid area. Solid tumors may be benign (non-cancer), or malignant (cancer). Different types of malignant solid tumors are named after the cell type that forms them. Examples of malignant solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (blood cancer) does not form malignant solid tumors generally. Malignant solid tumors include but are not limited to the abnormal cell mass that may be derived from different tissue types, such as liver, colon, colorectal, skin, breast, pancreas, cervix uteri, uterine body, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testis, thyroid or lung etc., so malignant solid tumors include malignant solid liver tumors, colon tumors, colorectal tumors, skin tumors, breast tumors, pancreatic tumors, cervix tumors, uterine body tumors, bladder tumors, gallbladder tumors, kidney tumors, laryngeal tumors, lip tumors, oral cavity tumors, esophageal tumors, ovarian tumors, prostate tumors, stomach tumors, testicular tumors, thyroid tumors or lung tumors etc.

[0155] Thus, in a more preferred embodiment of the present invention, the solid tumor is selected from tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestine (colon cancer, rectal cancer), pelvic cavity (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer and bladder, etc.

[0156] 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.

[0157] In the present invention, for the purpose of treatment, the term "subject" is preferably a subject in need of treatment for a target pathological condition, such as a tumor. For the purpose of prevention, the subject is preferably a subject at risk of developing a target pathological condition or prone to developing a target pathological condition. The term "subject" includes living organisms, such as prokaryotes and eukaryotes. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, hedgehogs, rats, and transgenic non-human animals. In a specific embodiment of the present invention, the subject is a human.

[0158] As used herein, "treatment" is a process for obtaining a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing the proliferation of neoplastic or cancerous cells (or destroying neoplastic or cancerous cells), inhibiting the metastasis of neoplastic cells, shrinking or reducing the size of a tumor, alleviating a malignant tumor, alleviating symptoms caused by a malignant tumor, improving the quality of life of a subject suffering from a malignant tumor, reducing the dosage of other drugs required to treat a malignant tumor, delaying the progression of a malignant tumor, curing a malignant tumor, and / or prolonging the survival of a patient suffering from a malignant tumor.

[0159] As used herein, an "effective amount" or "effective dose" of a bacterium, drug, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes that present during the course of disease. For therapeutic uses, beneficial or desired results include, for example, alleviating one or more symptoms of a disease (such as a tumor), reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, prolonging the survival of the treated subject, and / or delaying the progression of cancer in a patient. For example, an "effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in animal model systems that are predictive of efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by assays well known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Such an amount can be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0160] In some embodiments of the invention, the effective amount of bacteria in the bacteria, medicament or pharmaceutical composition comprises at least about 10 4 colony forming units (cfu), for example at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13cfu, including the ranges between any of the listed values, such as 10 4 -10 8 cfu, 10 4 -10 9 cfu, 10 4 -10 10 cfu, 10 4 -10 11 cfu, 10 4 -10 12 cfu, 10 4 -10 13 cfu, 10 5 -10 8 cfu, 10<00000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​cfu or 10 8 -10 13 cfu.

[0161] In some embodiments of the invention, the volume of an effective dose of the bacterium, medicament or pharmaceutical composition of the invention is less than or equal to about 3 ml, about 2.5 ml, about 2 ml, about 1.5 ml, about 1 ml, about 0.75 ml, about 0.5 ml, about 0.25 ml or about 0.1 ml. In some embodiments, the volume of an effective dose of the bacterium, medicament or pharmaceutical composition is about 20 ml, about 19 ml, about 18 ml, about 17 ml, about 16 ml, about 15 ml, about 14 ml, about 13 ml, about 12 ml, about 11 ml, about 10 ml, about 9 ml, about 8 ml, about 7 ml, about 6 ml, about 5 ml, about 4 ml, about 3 ml, about 2 ml or about 1 ml. Optionally, the volume of the effective dose of the bacteria, drug or pharmaceutical composition is about 20.5 ml, about 19.5 ml, about 18.5 ml, about 17.5 ml, about 16.5 ml, about 15.5 ml, about 14.5 ml, about 13.5 ml, about 12.5 ml, about 11.5 ml, about 10.5 ml, about 9.5 ml, about 8.5 ml, about 7.5 ml, about 6.5 ml, about 5.5 ml, about 4.5 ml, about 3.5 ml, about 2.5 ml, about 1.5 ml, or about 0.5 ml. In some embodiments, the volume of an effective dose of the bacteria, drug or pharmaceutical composition is about 200 ml, about 190 ml, about 180 ml, about 170 ml, about 160 ml, about 150 ml, about 140 ml, about 130 ml, about 120 ml, about 110 ml, about 100 ml, about 90 ml, about 80 ml, about 70 ml, about 60 ml, about 50 ml, about 40 ml or about 30 ml, as well as ranges between any of the listed values, for example, 100 ml-110 ml, 100 ml-120 ml, 90 ml-120 ml, 90 ml-130 ml, etc. Optionally, the volume of the effective dose of the bacteria, drug or pharmaceutical composition is about 205 ml, about 195 ml, about 185 ml, about 175 ml, about 165 ml, about 155 ml, about 145 ml, about 135 ml, about 125 ml, about 115 ml, about 105 ml, about 95 ml, about 85 ml, about 75 ml, about 65 ml, about 55 ml, about 45 ml, about 35 ml or about 25 ml, and ranges between any of the listed values, such as 105 ml-115 ml, 105 ml-125 ml, 95 ml-125 ml, 95 ml-135 ml, etc.Optionally, the volume of an effective dose of the bacteria, drug or pharmaceutical composition is about 900 microliters, about 800 microliters, about 700 microliters, about 600 microliters, about 500 microliters, about 400 microliters, about 300 microliters, about 200 microliters, or about 100 microliters, optionally about 950 microliters, about 850 microliters, about 750 microliters, about 650 microliters, about 550 microliters, about 450 microliters, about 350 microliters, about 250 microliters, about 150 microliters, or about 50 microliters.

[0162] In some embodiments, the effective dose of the bacterium, drug, or pharmaceutical composition has a volume of less than or equal to about 2.0 ml.

[0163] The dosage ranges mentioned herein are merely exemplary and do not limit the dosage ranges that can be selected by a physician. The amount of the active ingredient (e.g., the bacteria of the present invention) in the pharmaceutical compositions of the present invention can vary depending on factors such as the individual's disease state, age, sex, and weight, including the presence or absence of a tumor, the type of tumor being treated, the severity of the tumor, the activity or viability of the bacteria, drug, or pharmaceutical composition, the route of administration, the duration of treatment, the drugs used in combination with the bacteria, drug, or pharmaceutical composition (if any), the subject's diet and general health, and similar factors well known in the art. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, a single dose can be administered, several divided doses can be administered over time, or the dosage can be proportionally reduced or increased depending on the urgency of the therapeutic situation.

[0164] As used herein, a "pharmaceutically acceptable carrier" includes any material that allows the ingredient to retain biological activity when combined with the active ingredient and does not react with the subject's immune system, including but not limited to disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants, or lubricants. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion). For example, depending on the route of administration, the bacteria of the present invention can be wrapped in a material to protect the bacteria from the effects of acids and other natural conditions that can inactivate the bacteria. Pharmaceutically acceptable carriers include physiological saline, PBS buffer, sterile aqueous solutions or dispersions, and powders for the temporary preparation of injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any incompatibility with the active compound, may be in the pharmaceutical composition of the present invention.

[0165] Therefore, another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of the expression vector of the present invention or the modified bacteria of the present invention. In one embodiment, the modified bacteria are live bacteria.

[0166] In a preferred embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0167] 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.

[0168] In a preferred embodiment of the present invention, the pharmaceutical composition is used to treat solid tumors. The bacteria, drugs, or pharmaceutical compositions of the present invention are administered by intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0169] 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.

[0170] The dosage form of the drug or pharmaceutical composition of the present invention can be a solution, emulsion, freeze-dried preparation or suspension; for oral administration, the dosage form can be a tablet or capsule; for intranasal dosage form, the dosage form can be a powder, nasal drops or aerosol form; for topical administration, the dosage form can be an aqueous solution, suspension, ointment, cream or gel; for rectal or vaginal administration, the dosage form can be a suppository, enema or delivered as part of an endoscopic or colonoscopy procedure.

[0171] Bacteria, medicine or pharmaceutical compositions of the present invention can be manufactured by methods well known in the art, such as growing the microorganisms in a fermentation tank, followed by centrifugal concentration and washing, filtering or dialysis, conventional granulation, mixing, dissolving, encapsulating, lyophilizing or emulsifying processes and other methods. Bacteria, medicine or pharmaceutical compositions of the present invention can be produced in various forms, including particles, precipitations or microparticles, powders, including freeze-dried, rotary-dried or spray-dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. Preparations can optionally contain stabilizers, pH adjusting agents, surfactants, bioavailability regulators and combinations thereof.

[0172] Bacteria, medicine or pharmaceutical composition of the present invention can be used alone or in combination with other compounds or compositions in the presence of a carrier. In a preferred embodiment of the present invention, the bacterium, medicine or pharmaceutical composition can be used in combination with other malignant tumor therapies (including but not limited to, radiotherapy, chemotherapy and surgical operation). The bacterium, medicine or pharmaceutical composition can be used as an adjuvant in therapy in such a case.

[0173] In another aspect, the present invention provides a method for treating tumors, comprising administering the expression vector of the present invention, the Gram-negative bacteria of the present invention, or the pharmaceutical composition of the present invention to a subject.

[0174] In one embodiment, the expression vector, Gram-negative bacteria, or pharmaceutical composition is used to treat tumors. In one embodiment, the expression vector, Gram-negative bacteria, or pharmaceutical composition is used to induce an anti-tumor specific immune response in a subject with a tumor. In one embodiment, the expression vector, Gram-negative bacteria, or pharmaceutical composition is used to induce anti-tumor immune memory in a subject with a tumor. In one embodiment, the expression vector, Gram-negative bacteria, or pharmaceutical composition is used to prevent or treat metastasis or recurrence of a tumor. In one embodiment, the expression vector, Gram-negative bacteria, or pharmaceutical composition is used to treat a tumor that has become resistant to or failed previous anti-tumor therapy.

[0175] In one embodiment of the present invention, the tumor is a tumor of the nervous system, respiratory system, digestive system, urinary system, reproductive system, hematopoietic system, lymphatic system, endocrine system, or skin and mucosa. In one embodiment, the tumor is a sarcoma or carcinoma. In one embodiment, the tumor is a solid tumor.

[0176] In a further preferred embodiment of the present invention, the solid tumor is selected from the group consisting of breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal (colon cancer, rectal cancer), pelvic (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer and bladder tumors / cancers, etc.

[0177] In a more preferred embodiment of the present invention, the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

[0178] The method for treating tumors provided by the present invention comprises administering an effective amount of the expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor.

[0179] In an embodiment of the present invention, the expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention can be administered by the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, topical administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0180] In some embodiments of the present invention, the expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention can be administered, for example, at intervals of about 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 hours, 1 day, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, including ranges between any two of the listed values, for example, 1 minute to 10 minutes, 1 minute to 30 minutes, 1 minute to 1 hour, 1 minute to 2 hours, 1 minute to 4 hours, 1 minute to 12 hours, 1 minute to 18 hours, or 1 minute to 24 hours. 1 day, 10 minutes - 30 minutes, 10 minutes - 1 hour, 10 minutes - 2 hours, 10 minutes - 4 hours, 10 minutes - 12 hours, 10 minutes - 18 hours, 10 minutes - 1 day, 30 minutes - 1 hour, 30 minutes - 2 hours, 30 minutes - 4 hours, 30 minutes - 12 hours, 30 minutes - 18 hours, 30 minutes - 1 day, 30 minutes - 2 days, 1 hour - 2 hours, 1 hour - 4 hours, 1 hour - 12 hours, 1 hour - 18 hours, 1 hour - 1 day, 4 hours - 12 hours, 4 hours - 18 hours, 4 hours - 1 day, 1 day - 2 days, 1 day - 3 days, 1 day - 4 days, 1 day - 5 days, 1 day - 7 days, 1 day - 10 days, 2 days - 3 days, 2 days - 4 days, 2 days - 5 days, 2 days - 7 days, 2 days - 10 days, or 5 days - 10 days. In some embodiments, the expression vector, Gram-negative bacteria, or pharmaceutical composition is administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks, or once every twenty-six weeks.In some embodiments, the expression vector, Gram-negative bacteria, or pharmaceutical composition is formulated to, for example, be expressed in about 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1 hour, 2, 3, 1 minute to 10 minutes, 1 minute to 30 minutes, 1 minute to 1 hour, 1 minute to 2 hours, 1 minute to 4 hours, 1 minute to 12 hours, 1 minute to 18 hours, 1 minute to 1 day, 10 minutes to 30 minutes, 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 4 hours, 10 minutes to 12 hours, 1 minute to 18 hours, 1 minute to 1 day, 10 minutes to 30 minutes, 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 4 hours, 10 minutes to 12 hours, 10 minutes - 18 hours, 10 minutes - 1 day, 30 minutes - 1 hour, 30 minutes - 2 hours, 30 minutes - 4 hours, 30 minutes - 12 hours, 30 minutes - 18 hours, 30 minutes - 1 day, 30 minutes - 2 days, 1 hour - 2 hours, 1 hour - 4 hours, 1 hour - 12 hours, 1 hour - 18 hours, 1 hour - 1 day, 4 hours - 12 hours, 4 hours - 18 hours, 4 hours - 1 day, 1 day - 2 days, 1 day - 3 days, 1 day - 4 days, 1 day - 5 days, 1 day - 7 days, 1 day - 10 days, 2 days - 3 days, 2 days - 4 days, 2 days - 5 days, 2 days - 7 days, 2 days - 10 days, or 5 days - 10 days. In some embodiments, the vector, bacteria, drug or pharmaceutical composition is formulated to be administered once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks or once every twenty-six weeks. The dosage regimen may depend on the pattern of pharmacokinetic decay that the practitioner expects to achieve. The progress of the therapy can be monitored by conventional techniques and assays. The dosage regimen can vary over time.

[0181] The present invention also provides a method for inducing an anti-tumor specific immune response in a subject having a tumor, comprising administering an effective amount of the expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to the subject having a tumor.

[0182] The present invention also provides a method for inducing anti-tumor immune memory in a subject having a tumor, comprising administering an effective amount of the expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to the subject having a tumor.

[0183] The present invention also provides a method for preventing or treating tumor metastasis or recurrence, comprising administering an effective amount of the expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor.

[0184] The present invention also provides a method for preventing or treating tumor metastasis or recurrence, comprising administering an effective amount of the expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from tumor metastasis or recurrence or a subject at high risk of tumor metastasis or recurrence.

[0185] The present invention also provides a method for treating a tumor that has become resistant to or failed to be treated with previous anti-tumor therapy, comprising administering an effective amount of the expression vector, Gram-negative bacteria, or pharmaceutical composition of the present invention to a subject suffering from a tumor that has become resistant to or failed to be treated with previous anti-tumor therapy.

[0186] The present invention achieves the following beneficial effects by adopting the above technical solution:

[0187] 1. The present invention combines a phage-derived promoter with a specific phage RNA polymerase gene to coordinate the two, and uses a plasmid-based expression platform to achieve stable and efficient expression of drug proteins in the tumor area.

[0188] 2. The present invention combines a phage-derived promoter with a membrane-permeabilizing protein gene and a drug protein gene to coordinate the three, thereby stably and efficiently delivering drugs and achieving efficient killing of tumor cells.

[0189] 3. The present invention rationally designs a hypoxia-specific gene expression cassette, which reduces the overall mutation rate of the modified bacteria, achieves specific distribution within the tumor, and accelerates clearance in normal organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0190] Figure 1. Agarose gel electrophoresis identification and verification of the positive hypoxia promoter yhbU-S and ynfK-S clones.

[0191] Figure 2. Amplification verification diagram of essential gene library identified by agarose gel electrophoresis.

[0192] Figure 3 Agarose gel electrophoresis identification of reverse hyperoxia promoter cyoA-S and ydcI-S clone verification.

[0193] Figure 4 Schematic diagram of the hypoxia-specific gene expression cassette.

[0194] Figure 5 Schematic diagram of the lambda RED recombinase and CRE recombinase systems.

[0195] Figure 6 shows the knockout experiment and identification of the Salmonella aroA gene.

[0196] (A) Agarose gel electrophoresis to identify the PCR amplification product of the aroA gene knockout fragment;

[0197] (B) Verification of the PCR amplification product of the target fragment of strain SWT003 identified by agarose gel electrophoresis.

[0198] Figure 7 shows the knockout experiment and identification of the Salmonella alr gene.

[0199] (A) Agarose gel electrophoresis to identify the PCR amplification product of the alr gene knockout fragment;

[0200] (B) Verification of the PCR amplification product of the target fragment of strain SWT004 identified by agarose gel electrophoresis.

[0201] FIG8 is a graph showing the verification of strains SWT1001 and SWT1005 after insertion of the hypoxia-specific gene expression cassette (alr is an essential gene) as identified by agarose gel electrophoresis.

[0202] FIG9 is a diagram showing the verification of the PCR amplification product of SWT007 identified by agarose gel electrophoresis.

[0203] FIG10 is a graph showing the verification of strains SWT2009 and SWT2013 after insertion of the hypoxia-specific gene expression cassette (dadX is an essential gene) as identified by agarose gel electrophoresis.

[0204] FIG11 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1001.

[0205] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0206] (B) Aerobic culture on LB plates without D-alanine.

[0207] FIG12 shows the results of the oxygen adaptability verification test of Salmonella strain SWT1005.

[0208] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0209] (B) Aerobic culture on LB plates without D-alanine.

[0210] FIG13 shows the results of the oxygen adaptability verification test of Salmonella strains SWT2009 and SWT2013.

[0211] (A) SWT2009 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0212] (B) SWT2013 was cultured on LB plates without D-alanine. The upper plate was cultured under anaerobic conditions, while the lower plate was cultured under aerobic conditions.

[0213] FIG14 shows the results of the oxygen adaptability verification test of various Salmonella strains at an oxygen concentration below 0.8%.

[0214] (A) Salmonella strain SWT1001 was cultured on LB plates;

[0215] (B) Salmonella strain SWT1005 was cultured on LB plates;

[0216] Figure 15 is a schematic diagram of the pPRO005 cassette containing the chloramphenicol resistance gene, lacUV5 and T7 RNA polymerase genes.

[0217] Figure 16: Enzyme digestion identification of plasmid pPRO005.

[0218] The arrow points to the excised T7 RNA polymerase gene cassette fragment.

[0219] Figure 17 shows the identification of the recombination of the T7 RNA polymerase constitutive expression cassette controlled by lacUV5 into the Salmonella chromosome.

[0220] (A) PCR amplified fragment used for recombination (primers SWTO102 and SWTO103);

[0221] (B) PCR identification diagram of strains SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P after construction.

[0222] Figure 18 is a schematic diagram of the construction of plasmid expression vector pPRO010.

[0223] Figure 19 shows PCR identification of plasmid pPRO010 in strains SWT5001, SWT5005, SWT6009 and SWT6013.

[0224] Figure 20 is a schematic diagram of the construction of plasmid expression vector pPRO012.

[0225] Figure 21 shows PCR identification of plasmid pPRO012 in strains SWT7001, SWT7005, SWT7009 and SWT7013.

[0226] Figure 22 Western Blot identification of the expression of target genes in the expression vector strain and the control vector strain under culture medium conditions.

[0227] (A) Expression of DTA in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013;

[0228] (B) Expression of LLO in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013;

[0229] (C) Expression of T7 RNA polymerase in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013.

[0230] FIG23 Western Blot identification of target gene expression in mouse tumors using expression vector strains and control vector strains.

[0231] (A) Expression of DTA in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013;

[0232] (B) Expression of LLO in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013;

[0233] (C) Expression of T7 RNA polymerase in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013.

[0234] Figure 24 shows identification of mouse tumor sections after treatment with strain SWT5005.

[0235] The dark part indicated by the arrow is the antibody-positive staining area;

[0236] (A) Tumor sections stained with anti-Salmonella antibodies;

[0237] (B) Tumor sections stained with anti-HIF1α antibody;

[0238] (C) Tumor sections stained with anti-Diphtheria toxin antibody.

[0239] Figure 25 shows the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0240] (A) EMT6;

[0241] (B)K7M2;

[0242] (C)Hepa1-6.

[0243] Figure 26 shows the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0244] (D)A549;

[0245] (E)B16F10;

[0246] (F)Renca.

[0247] Figure 27 shows the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0248] (G)MFC;

[0249] (H)Pan02;

[0250] (I)RM-1.

[0251] Figure 28 shows the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0252] (J)CT26;

[0253] (K) ID8;

[0254] (L) Neuro-2a.

[0255] Figure 29 shows the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0256] (M)SCC7;

[0257] (N)MB49.

[0258] Figure 30 shows the killing effect of CCK8-identified expression vector strains and control vector strains on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7 and MB49 cancer cells.

[0259] (A) SWT5001 and SWT7001;

[0260] (B) SWT5005 and SWT7005.

[0261] Figure 31 shows the killing effects of CCK8-identified expression vector strains and control vector strains on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7 and MB49 cancer cells.

[0262] (A) SWT6009 and SWT7009;

[0263] (B) SWT6013 and SWT7013.

[0264] Figure 32 shows the identification of the inhibitory effects of expression vector strain SWT5005 and control vector strain SWT7005 in different mouse tumor models.

[0265] Vehicle: blank control, no drug treatment;

[0266] (A) EMT6;

[0267] (B)K7M2;

[0268] (C) Hepa1-6;

[0269] (D)A549.

[0270] Figure 33 shows the identification of the inhibitory effects of expression vector strain SWT5005 and control vector strain SWT7005 in different mouse tumor models.

[0271] Vehicle: blank control, no drug treatment;

[0272] (A)B16F10;

[0273] (B) Renca;

[0274] (C)MFC;

[0275] (D)Pan02.

[0276] Figure 34 illustrates the identification of the inhibitory effects of expression vector strain SWT5005 and control vector strain SWT7005 in different mouse tumor models.

[0277] Vehicle: blank control, no drug treatment;

[0278] (A)RM-1;

[0279] (B) CT26;

[0280] (C) ID8;

[0281] (D) Neuro-2a.

[0282] Figure 35 shows the identification of the inhibitory effects of expression vector strain SWT5005 and control vector strain SWT7005 in different mouse tumor models.

[0283] Vehicle: blank control, no drug treatment;

[0284] (A) SCC7;

[0285] (B)MB49. DETAILED DESCRIPTION

[0286] The present application will be further described in detail below through the accompanying drawings and examples, through which the features and advantages of the present application will become more clear and distinct.

[0287] Example 1: Construction of a hypoxia-specific gene expression cassette with alr and dadX genes as essential genes for survival

[0288] In this example, the forward hypoxia promoter used Salmonella yhbU (yhbU-S) (SEQ ID No. 31), the survival essential gene used Salmonella alr gene (SEQ ID No. 27), the reverse hyperoxia promoters used Salmonella cyoA (cyoA-S) (SEQ ID No. 33) and Salmonella ydcI (ydcI-S) (SEQ ID No. 34), and the forward hypoxia promoter used Escherichia coli ynfK (ynfK-E) (SEQ ID No. 32), the survival essential gene used Salmonella dadX gene (SEQ ID No. 29), and the reverse hyperoxia promoters used Salmonella cyoA (cyoA-S) (SEQ ID No. 33) and Salmonella ydcI (ydcI-S) (SEQ ID No. 34) to construct a hypoxia-specific gene expression cassette.

[0289] The combination of hypoxia-specific gene expression cassettes is shown in Table 1.

[0290] Table 1 Combinations of forward hypoxia promoter and reverse hyperoxia promoter

[0291] (The essential genes for survival are the alr gene or dadX gene of Salmonella)

[0292] (I) Construction of a positive hypoxia promoter clone library

[0293] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Industrial Microbiological Culture Collection) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) and inoculate it into 5 ml LB liquid medium and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The OD 600 The reading is between 2-3;

[0294] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium and mix it with the corresponding primers and high-fidelity PCR amplification enzyme (purchased from TAKARA) system, and then put the mixture into the PCR amplification device;

[0295] 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;

[0296] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0297] 4. Mix the recovered product with the endonuclease (purchased from NEB) digestion system and incubate at 37°C for 1 hour;

[0298] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0299] The primers and endonucleases used for different forward hypoxia promoters are as follows:

[0300] 1. yhbU-S (SEQ ID No. 31): strain SWT001 was amplified using primers SWTO19 and 20, and the product was double-cleaved with NotI and HindIII and recovered;

[0301] 2. ynfK-E (SEQ ID No. 32): amplified from E. coli strain DH10B using primers SWTO21, 22, and the product was double-cleaved with NotI and HindIII and recovered;

[0302] The results of verification of the forward hypoxia promoter clone library by agarose gel electrophoresis are shown in FIG1 .

[0303] (2) Construction of a gene library essential for survival

[0304] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC, China Industrial Microbiology Culture Collection) and inoculate it into 5 ml of LB liquid medium. Incubate the culture in a shaker at 37°C and 220 rpm for 16 hours.

[0305] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium, mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then place the mixture into the PCR amplification device;

[0306] 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;

[0307] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0308] 4. Mix the recovered product with the enzyme digestion system and incubate at 37 degrees for 1 hour;

[0309] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0310] The essential genes for survival from different sources are as follows:

[0311] 1. Salmonella alr gene (SEQ ID No. 27): strain SWT001 was amplified using primers SWTO35 and 36, and the product was double-cut with HindIII and XhoI and recovered;

[0312] 2. Salmonella dadX gene (SEQ ID No. 29): strain SWT001 was amplified using primers SWTO39 and 40, and the product was double-cut with HindIII and XhoI and recovered;

[0313] The results of verification of the essential survival gene library by agarose gel electrophoresis are shown in FIG2 .

[0314] (III) Construction of the reverse hyperoxia promoter library

[0315] 1. Pick a single colony of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC, China Industrial Microbiology Culture Collection) and inoculate it into 5 ml of LB liquid medium. Incubate it in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0316] 2. Use a pipette tip to draw 2 μl of the corresponding strain culture medium, mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then place the mixture into the PCR amplification device;

[0317] 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;

[0318] 3. The amplified PCR product is purified and recovered through a DNA gel recovery system;

[0319] 4. Mix the recovered product with the enzyme digestion system and incubate at 37°C for 1 hour;

[0320] 5. Purify and recover the enzyme-digested products through a DNA gel recovery system.

[0321] The primers and endonucleases used for different reverse hyperoxia promoters are as follows:

[0322] 1. cyoA-S (SEQ ID No. 33): strain SWT001 was amplified using primers SWTO43 and 44, and the product was double-cleaved with XhoI and PstI and recovered;

[0323] 2. ydcI-S (SEQ ID No. 34): strain SWT001 was amplified using primers SWTO51 and 52, and the product was double-cut with XhoI and PstI and recovered;

[0324] The results of verifying the reverse high oxygen promoter clone library by agarose gel electrophoresis are shown in FIG3 .

[0325] (IV) Construction of a combinatorial library of forward hypoxia promoters, essential survival genes, and reverse hyperoxia promoters

[0326] The composition of the hypoxia-specific gene expression cassette is shown in FIG4 , which is composed of a forward hypoxia promoter, a survival-essential gene, and a reverse hyperoxia promoter in sequence.

[0327] As shown in Table 1, a cloned library was formed by combining sequences of the forward hypoxia promoter library digested with NotI and HindIII, the survival essential gene Salmonella alr gene (SEQ ID No. 27) or Salmonella dadX gene (SEQ ID No. 29) digested with HindIII and XhoI, and the reverse hyperoxia promoter library digested with XhoI and PstI, and ligated into the plasmid pSWT003 vector (vector backbone, pBlueScript SK (+), purchased from Biowind) digested with SpeI and PstI, and the plasmid pSWT007 vector (containing double-sided same-direction loxP sequences and a DNA fragment of the chloramphenicol resistance gene (SEQ ID No. 37)) digested with SpeI and NotI.

[0328] The products amplified from Salmonella SWT001 or Escherichia coli DH10B using the above primers and recovered by enzyme digestion were combined and ligated by enzyme ligation reaction, transformed into DH10B bacteria and coated on LB plates containing 25 μg / ml chloramphenicol to obtain the corresponding plasmids shown in Table 1.

[0329] (V) Construction of the strain corresponding to the present invention

[0330] 1. Wild-type Salmonella Typhimurium (SWT001)

[0331] Wild-type Salmonella typhimurium (SWT001) was purchased from China Industrial Microbiology Culture Collection Center (CICC).

[0332] 2. Construction of Salmonella (SWT002) containing temperature-inducible lambda-RED recombinase and loxp-CRE enzyme systems

[0333] 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.

[0334] Plasmid pSWT001, shown in Figure 5, contains a lambda-RED recombinase module (SEQ ID No. 38) (similar in function to the plasmid vector psim6 from Biowind) and a loxP-Cre recombinase module (SEQ ID No. 38) (similar in function to the plasmid vector 705-Cre from Gene Bridges). The lambda-RED recombinase module consists of three recombinases: EXO, BET, and GAM. These recombinases are controlled by the CI857 temperature regulator. Therefore, expression is lost at 32°C and is only activated at temperatures above 37°C. Under temperature-induced conditions, the recombinase EXO cleaves the 5' end of double-stranded linear DNA, creating a single-stranded DNA overhang at the 3' end. This single-stranded DNA is bound by the BET protein and protected from degradation by other nucleases. GAM inhibits endogenous bacterial nucleases. Homologous arms for homologous recombination are approximately 35-50 bp and are added by PCR to flank the DNA fragment to be recombined. The advantage of this technology is that it can accurately target the target area of ​​the bacterial chromosome without causing additional mutations. The outermost parts of the recombinant double-stranded DNA fragments include homologous arm sequences of the position where recombination is required, which contain loxp sequences in the same direction on both sides, totaling 34bp. The sites of the double loxp in the same direction are chloramphenicol resistance genes, which are used to screen recombinant bacteria. After successful recombination, the CRE enzyme system carried on the pSWT001 plasmid (also controlled by the CI857 temperature regulator) specifically recognizes the loxp sequence and can cut the sequence in the middle of the loxp in the same direction, leaving a loxp sequence, thereby eliminating the chloramphenicol resistance gene. Repeating the above operations can achieve continuous knockout and knock-in of genes.

[0335] The specific steps are as follows:

[0336] (1) Streak the strain SWT001 on an LB plate and culture it in a 37°C incubator overnight;

[0337] (2) Pick a single clone and inoculate it into 5 ml of LB liquid medium and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0338] (3) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3 Place on ice and let stand for 1 hour;

[0339] (4) Wash the cells three times with sterilized purified water;

[0340] (5) The recovered cells were mixed with 10 ng of plasmid pSWT001 and electroporated at a voltage of 1.8 kV;

[0341] (6) The electroporated cells were spread on LB plates containing 100 μg / ml ampicillin sodium and cultured overnight in a 32°C constant temperature incubator until a single clone colony was grown, named SWT002.

[0342] 3. Construction of attenuated Salmonella with aroA gene knockout (SWT003)

[0343] (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 cultured in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0344] (2) Inoculate the culture into fresh LB liquid medium containing 100 μg / ml ampicillin sodium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0345] (3) Wash the cells three times with sterilized purified water.

[0346] (4) Prepare PCR products of SWTO1 and SWTO2.

[0347] Primers SWTO1 and SWTO2 were mixed with plasmid pSWT002 (containing loxP sequences in the same direction on both sides and a chloramphenicol resistance gene SEQ ID No. 39 in the middle), and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0348] 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.

[0349] The amplified PCR products were subjected to agarose gel electrophoresis to verify the PCR amplification products of SWTO1 and SWTO2 of the aroA gene knockout fragments. The verification results are shown in Figure 6A.

[0350] The PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were measured using nanodrop.

[0351] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO1 and SWTO2 and electroporated at a voltage of 1.8 kV.

[0352] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0353] (7) Positive clones were identified by colony PCR, and the insertion of chloramphenicol resistance gene was identified using SWTO4, SWTO6, SWTO3 and SWTO5.

[0354] The target fragment PCR amplification product of strain SWT003 was verified by agarose gel electrophoresis, and the verification results are shown in Figure 6B.

[0355] (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.

[0356] 4. Construction of alr gene knockout attenuated bacteria (SWT004)

[0357] (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.

[0358] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0359] (3) Wash the cells three times with sterilized purified water.

[0360] (4) Preparation of PCR products of SWTO70 and SWTO71.

[0361] Primers SWTO70 and SWTO71 were mixed with plasmid pSWT002 and a high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0362] 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.

[0363] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0364] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO70 and SWTO71 and electroporated at a voltage of 1.8 kV.

[0365] The PCR amplification products of SWTO70 and SWTO71 of the alr gene knockout fragments were verified by agarose gel electrophoresis. The verification results are shown in Figure 7A.

[0366] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0367] (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.

[0368] The PCR amplification product of the target fragment of strain SWT004 was verified by agarose gel electrophoresis, and the verification results are shown in Figure 7B (the primers used were SWTO72, SWTO5, SWTO6, and SWTO73).

[0369] (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.

[0370] Example 2: Integration of the hypoxia-specific gene expression cassette into the dadX gene locus on the chromosome of the SWT004 strain (destroying its function)

[0371] Construction of alr-deficient attenuated bacteria containing a hypoxia-specific gene expression cassette

[0372] 1. Pick a single clone of the alr gene-knockout attenuated bacteria SWT004 and inoculate it into 5 ml of fresh LB liquid culture medium. Place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0373] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue to culture for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0374] 3. Wash the bacteria 3 times with sterilized purified water.

[0375] 4. Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette. Mix primers SWTO78 and SWTO79 with the hypoxia-specific gene expression cassette plasmids pOL1001 and pOL1005, and a high-fidelity PCR amplification enzyme system and place them in a PCR amplification device.

[0376] 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.

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

[0378] 5. Mix the recovered bacteria with 100 ng of PCR product and electroporate at a voltage of 1.8 kV.

[0379] 6. The electroporated cells were spread on plates containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine and cultured overnight in a 32°C constant temperature incubator until single clones were grown. The resulting strains were SWT1001 and SWT1005.

[0380] 7. Inoculate the single clone into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours to allow the CRE enzyme to act and eliminate the chloramphenicol resistance gene.

[0381] 8. Using primers SWTO61, 20; SWTO75, 20, clones were identified by colony PCR. The identification results of strain SWT1001 (yhbU-S+alr+cyoA-S combination) and strain SWT1005 (yhbU-S+alr+ydcI-S combination) are shown in Figure 8.

[0382] Example 3: Construction of a hypoxia-specific gene expression cassette with dadX gene as an essential gene for survival

[0383] Since alanine racemase has two isoenzyme genes in the genomes of bacteria such as Salmonella and Escherichia coli, namely the alr gene and the dadX gene, to verify the effect, the essential genes for survival of some of the hypoxia-specific gene expression cassettes were replaced with the corresponding homologous genes dadX genes pOL2009 and pOL2013.

[0384] The hypoxia-specific gene expression cassette was integrated into the alr gene locus on the chromosome of the SWT007 strain.

[0385] 1. Construction of dadX gene knockout attenuated bacteria (SWT007)

[0386] (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.

[0387] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density reaches OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0388] (3) Wash the cells three times with sterilized purified water.

[0389] (4) Preparation of PCR products of SWTO78 and SWTO79.

[0390] Primers SWTO78 and SWTO79 were mixed with plasmid pSWT002 (same as above, plasmid description), and high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0391] 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.

[0392] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0393] (5) The recovered cells were mixed with 100 ng of PCR products of SWTO78 and SWTO79 and electroporated at a voltage of 1.8 kV.

[0394] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured overnight in a constant temperature incubator at 32°C until a single clone colony was grown.

[0395] (7) Positive clones were identified by colony PCR, and SWTO59, SWTO5, SWTO6, and SWTO87 were used to identify the insertion of the chloramphenicol resistance gene, as shown in Figure 9.

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

[0397] 2. Construction of defective attenuated bacteria containing a hypoxia-specific gene expression cassette and the essential survival gene being the Salmonella dadX gene

[0398] (1) A single clone of the dadX gene-knockout attenuated bacteria SWT007 was selected and inoculated into 5 ml of LB liquid culture medium, and then placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

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

[0400] (3) Wash the cells three times with sterilized purified water.

[0401] (4) Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette. Mix primers SWTO85 and SWTO86 with the hypoxia-specific gene expression cassette plasmids pOL2009 and pOL2013, and a high-fidelity PCR amplification enzyme system and place them in a PCR amplification device.

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

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

[0404] (5) The recovered bacteria were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV.

[0405] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine and cultured overnight in a 32°C constant temperature incubator until a single clone colony was grown.

[0406] (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.

[0407] (8) Using the corresponding primers SWTO98, 22; SWTO99, 22, clones were identified by colony PCR. The identification results of strains SWT2013 (ynfK-E + dadX + ydcI-S) and strain SWT2009 (ynfK-E + dadX + cyoA-S) are shown in Figure 10.

[0408] Example 4: Verification of oxygen adaptability of Salmonella strains SWT1001, SWT1005, SWT2009, and SWT2013 containing a hypoxia-specific gene expression cassette

[0409] 1. Pick a single clone of strains SWT1001, SWT1005, SWT2009, and SWT2013 containing the hypoxia-specific expression cassette and inoculate it into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine. Place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0410] 2. After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0411] 3. Calculate the volume of bacterial solution per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0412] 4. Take the above bacterial solution The culture was performed on a plate labeled "1" and repeated three times, labeled "a", "b", and "c".

[0413] 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".

[0414] 6. Repeat this 10-fold gradient dilution until you reach the mark "8".

[0415] 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).

[0416] The results of the oxygen adaptability verification test of the strains containing the hypoxia-specific expression cassette are shown in Figures 11 to 13. The results show that the Salmonella strains SWT1001, SWT1005, SWT2009, and SWT1023 containing the hypoxia-specific gene expression cassette exhibited hypoxia regulation capabilities.

[0417] Example 5: Oxygen Concentration Simulation of a Strain Library Containing Hypoxia-Specific Gene Expression Cassettes

[0418] By using anaerobic gas production bags to consume the oxygen in the sealed culture tank, and using an oxygen meter to measure the oxygen concentration in the sealed culture tank, the oxygen concentration in the sealed culture tank can be fixed within a specific concentration range after using the anaerobic gas production bags for a certain period of time. The specific method is as follows:

[0419] The anaerobic gas production bag (brand: Japan Mitsubishi, product number: D-119) was placed in a 7.0 L sealed culture tank (brand: Japan Mitsubishi, product number: D-112) and an oxygen meter (brand: Meicheng Electrochemical, product number: OX-100A) was placed in the tank.

[0420] The above anaerobic gas production bag combination was used to verify the growth of the genetically modified strain under an oxygen concentration below 0.8%.

[0421] 1. Verification of the adaptability of the strain library containing hypoxia-specific gene expression cassettes to oxygen concentrations below 0.8%

[0422] Since the pathological hypoxic zone of a tumor is an oxygen concentration below 1%, the present invention intends to demonstrate that when the oxygen concentration is below 1% (or close to 1%), Salmonella transformed with the hypoxia-specific gene expression cassette can still grow normally.

[0423] Oxygen concentration control is accomplished by anaerobic gas production bags. After one hour of anaerobic gas production, the oxygen meter reading shows an oxygen concentration of 0.8-1%. Therefore, this experiment simulates the hypoxic environment within the tumor and verifies the growth of Salmonella on a petri dish after one hour of anaerobic gas production bags in a sealed culture jar. This also simulates the growth of Salmonella strains containing a hypoxia-specific gene expression cassette in the hypoxic environment within the tumor.

[0424] The operating procedures of the spot plate experiment are as follows:

[0425] (1) A single clone of the strain containing the hypoxia-specific expression cassette was selected and inoculated into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and cultured in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0426] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0427] (3) Calculate the volume of bacterial solution per OD of bacteria using the following formula and add deionized water to 1 ml.

[0428] (4) Take 10 μl of the above bacterial solution and spot it on an LB plate containing D-alanine for culture, mark it as "1" and repeat three times, mark it as "a", "b", and "c".

[0429] (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".

[0430] (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”.

[0431] (7) Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0432] (8) The two plates were placed in a 0.8% oxygen environment at 37°C and cultured.

[0433] As shown in Figure 14 , the present invention compared the growth of different strains (Figure 14(A) SWT1001, Figure 14(B) STW1005) on LB plates without D-alanine to determine the growth performance of each strain at oxygen concentrations below 0.8%. Because the culture medium without D-alanine requires the corresponding Salmonella strains to rely on their own oxygen regulation systems for normal growth, the results confirmed that strains containing the hypoxia-specific expression cassette, which can grow normally in an anaerobic environment, can also grow normally at oxygen concentrations below 0.8%.

[0434] Example 6: Construction of Salmonella strains (SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, SWT2013-T7P) with T7 RNA polymerase homologous replacement of the asd gene site

[0435] (1) Construction of lacUV5-controlled T7 RNA polymerase constitutive expression plasmid pPRO005

[0436] To construct a constitutively expressed T7 RNA polymerase, the vector pPRO005 was constructed. The process, as shown in Figure 15 , involves placing the constitutively expressed promoter lacUV5 upstream of the RBS sequence (SEQ ID No. 35) and T7 RNA polymerase (SEQ ID No. 4). Furthermore, the chloramphenicol resistance gene cm (SEQ ID No. 37) flanked by loxP sequences was added upstream of lacUV5. This facilitates recombination onto the Salmonella chromosome and facilitates screening.

[0437] 1. Plasmid pSWT007 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with SpeI and NotI to recover an approximately 1100 bp fragment containing the chloramphenicol resistance gene cm flanked by loxP sequences.

[0438] 2. Direct annealing of primers SWTO100 and SWTO101 to generate NotI and HindIII cuts and recover a fragment of approximately 70 bp, including the promoter lacUV5;

[0439] 3. Plasmid pPRO004 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with HindIII and XhoI to recover a 2690 bp fragment including the T7 RNA polymerase gene;

[0440] 4. Cut plasmid pSWT003 (plasmid backbone) with SpeI and XhoI to recover a fragment of approximately 3000 bp;

[0441] 5. The above five fragments were ligated using T4 DNA ligase, transformed into DH10B competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and coated on ampicillin and chloramphenicol double-resistant LB plates, and a single clone was screened and named pPRO005;

[0442] 6. Plasmid pPRO005 was extracted, and 100 ng was mixed with NotI and XhoI endonuclease system, incubated at 37°C for 1 hour, and identified by agarose gel electrophoresis, as shown in Figure 16.

[0443] (ii) The recombinant lacUV5-controlled T7 RNA polymerase constitutive expression cassette is integrated into the Salmonella chromosome

[0444] 1. Pick single clones from SWT1001, SWT1005, SWT2009, and SWT2013, respectively, and inoculate them into 5 ml LB liquid culture medium containing 100 μg / ml D-alanine. Place the culture medium in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0445] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue to culture for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place it on ice for 1 hour.

[0446] 3. Wash the bacteria 3 times with sterilized purified water.

[0447] 4. Prepare PCR products of SWTO102 and SWTO103.

[0448] Primers SWTO102 and SWTO103 were mixed with plasmid pPRO005 and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0449] 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.

[0450] The amplified PCR products were subjected to agarose gel electrophoresis to verify the PCR amplification products of SWTO102 and SWTO103. The verification results are shown in FIG17A .

[0451] The amplified PCR products were purified and recovered using a DNA gel recovery system, and the concentration and purity of the recovered PCR products were determined using nanodrop.

[0452] 5. The recovered bacteria were mixed with 100 ng of PCR products of SWTO102 and SWTO103 and electroporated at a voltage of 1.8 kV.

[0453] 6. Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol, 100 μg / ml D-alanine, and 100 μg / ml DAP (diaminopimelic acid), and culture in a 32°C incubator overnight until a single clone colony grows.

[0454] 7. Inoculate the single clone into 5 ml of LB medium containing 100 μg / ml DAP and 100 μg / ml D-alanine, and culture in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0455] 8. Identify positive clones by colony PCR, using SWTO104, SWTO106, SWTO105, and SWTO107 for identification.

[0456] The target fragment PCR amplification products of strains SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P were verified by agarose gel electrophoresis. The verification results are shown in Figure 17B.

[0457] Example 7: Construction of strains carrying the three-in-one expression vector pPRO010 (SWT5001, SWT5005, SWT6009, SWT6013)

[0458] In this example, a plasmid vector without antibiotic resistance selection was constructed, as shown in Figure 18. The pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were used as balanced lethality control mechanisms, and a T7 promoter containing a dual phage-derived promoter was used to control the expression of the drug protein exotoxin DTA fragment (SEQ ID No. 15) and the membrane-permeabilizing protein gene LLO (SEQ ID No. 13). The constructed plasmid pPRO010 was transformed into SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P, respectively, to generate the corresponding strains SWT5001, SWT5005, SWT6009, and SWT6013.

[0459] The specific steps are as follows:

[0460] 1. Plasmid pPRO006 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with XhoI and NotI to recover an approximately 820 bp fragment containing the pUC replicon.

[0461] 2. Plasmid pPRO007 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with PstI and XhoI to recover a 1270 bp fragment containing the Salmonella asd gene;

[0462] 3. The plasmid pPRO008 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with PstI to recover a 660 bp fragment containing the codon-optimized Salmonella gene DTA controlled by the T7 promoter;

[0463] 4. Plasmid pPRO009 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NotI and PstI to recover a 1660 bp fragment containing the codon-optimized Salmonella gene LLO controlled by the T7 promoter;

[0464] 5. The above four fragments were ligated by T4 DNA ligase and transformed into competent cells of SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P. The competent cells were coated with 100 μg / ml D-alanine LB plates and single clones were selected and named SWT5001, SWT5005, SWT6009, and SWT6013.

[0465] 6. Single clones were picked and amplified using primers SWTO108 and SWTO109; SWTO110 and SWTO111; SWTO112 and SWTO113; SWTO114 and SWTO115; SWTO116 and SWTO117; SWTO118 and SWTO119; SWTO120 and SWTO121; SWTO122 and SWTO123; SWTO124 and SWTO125; and identified by agarose gel electrophoresis, as shown in Figure 19.

[0466] Example 8: Construction of control strains (SWT7001, SWT7005, SWT7009, SWT7013) carrying the three-in-one expression vector pPRO012

[0467] As a control strain, the present invention constructed an expression vector that replaces the T7 promoter, using the promoter of the sseA gene on the pathogenicity island II of Salmonella (SEQ ID No. 40).

[0468] The sseA promoter is a strong promoter that can be induced to express after bacteria invade cells. In this example, as a control for the T7 promoter, a plasmid vector without antibiotic resistance selection was constructed. As shown in Figure 20, the pUC replicon (SEQ ID No. 36) and the Salmonella asd gene (SEQ ID No. 11) were used as balanced lethal control mechanisms, and the sseA promoter was used to control the expression of the drug protein exotoxin DTA fragment (SEQ ID No. 15) and the membrane-breaking protein gene LLO (SEQ ID No. 13). The constructed plasmid pPRO012 was respectively transferred into SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P, generating the corresponding strains SWT7001, SWT7005, SWT7009, and SWT7013.

[0469] The specific steps are as follows:

[0470] 1. Cut plasmid pPRO010 with PstI and NotI to recover a 2050 bp fragment containing the pUC replicon and asd gene;

[0471] 2. Plasmid pPRO008 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NdeI and PstI to recover a 590 bp fragment containing the codon-optimized Salmonella gene DTA.

[0472] 3. Plasmid pPRO009 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NdeI and PstI to recover a 1600 bp fragment containing the Salmonella codon-optimized gene LLO.

[0473] 4. Plasmid pPRO011 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was digested with NotI and NdeI to recover a 425 bp fragment containing the sseA promoter and RBS site sequence. This fragment served as the LLO promoter.

[0474] 5. Plasmid pPRO011 (synthesized by Beijing Liuhe BGI Genomics Co., Ltd.) was amplified using primers SWTO126 and SWTO127. A 425 bp fragment containing the sseA promoter and RBS site sequence was recovered and amplified using PstI and NdeI. This fragment served as the DTA promoter.

[0475] 6. The above five fragments were ligated with T4 DNA ligase and transformed into competent cells of SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P. The competent cells were then coated with 100 μg / ml D-alanine LB plates and single clones were selected and named SWT7001, SWT7005, SWT7009, and SWT7013.

[0476] 7. Single clones were picked and amplified using primers SWTO111 and SWTO128, and identified by agarose gel electrophoresis, as shown in Figure 21.

[0477] Example 9: Verification of the expression of T7 RNA polymerase, DTA, and LLO proteins in strains carrying expression vectors and control vectors

[0478] This example was designed to demonstrate that the protein regulated by the T7 promoter has stable expression in culture and tumor environments.

[0479] (I) Verification of protein expression in culture medium of expression vector strains SWT5001, SWT5005, SWT6009, SWT6013 and control vector strains SWT7001, SWT7005, SWT7009, SWT7013

[0480] 1. Streak strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 on LB plates supplemented with 100 μg / ml D-alanine, and culture them statically in a 37°C incubator overnight.

[0481] 2. Pick out single clones of strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 respectively, inoculate them into 5 ml LB liquid culture medium supplemented with 100 μg / ml D-alanine, and place them in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0482] 3. Take 1 ml of each strain's bacterial suspension, centrifuge at 12000 rpm, remove the supernatant, recover the precipitate, resuspend it in lysis buffer (protein loading buffer, purchased from CST, product number 7722S), heat it at 95°C for 10 minutes, centrifuge it again at 12000 rpm, and recover the supernatant for later use.

[0483] 4. Take 5 μl of the supernatant recovered in the previous step, add it to SDS-PAGE gel (SDS-PAGE gel preparation kit purchased from Sangon Biotechnology, product number C631100-0200), and perform electrophoresis at 120 V for 60 minutes.

[0484] 5. Transfer the SDS-PAGE gel after electrophoresis to a PVDF membrane (purchased from Roche, product number 03010040001) at a steady current of 250 mA for 30 minutes.

[0485] 6. Transfer the transferred PVDF membrane to 5% skim milk powder, place it on a horizontal shaker at 80 rpm, and incubate at room temperature for 1 hour as a blocking method.

[0486] 7. The blocked PVDF membrane was incubated with Anti-T7 RNA Polymerase antibody (T7 RNA polymerase antibody, purchased from Creative Biomart, Catalog No. CABT-B8990), Anti-diphtheria toxin antibody (DTA identification antibody, purchased from abcam, Catalog No. ab151222), and Anti-listeriolysin antibody (LLO identification antibody, purchased from abcam, Catalog No. ab200538) as primary antibodies diluted with 5% skim milk powder at 4°C for 18 hours (one antibody was used for each membrane).

[0487] 8. Use 5% skim milk powder to dilute the secondary antibody. Transfer the PVDF membrane after primary antibody incubation to the secondary antibody dilution solution and place it on a horizontal shaker at 80 rpm and incubate at room temperature for 1 hour.

[0488] 9. Wash the PVDF membrane after incubation with secondary antibody with TBST Buffer (purchased from Sangon Biotechnology, product number C520009-0005) for 3 times, 5 minutes each time. Add ECL developer (developer SignalFire TM ECL Reagent (CST, Cat. No. 6883p3) was purchased and incubated in the dark for 1 min. After incubation, the PVDF membrane was placed on a chemiluminescence instrument for image acquisition.

[0489] Under LB culture conditions, strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 identified by Western Blot can all express DTA at high intensity, see Figure 22A; LLO expression is shown in Figure 22B; T7 RNA polymerase expression is shown in Figure 22C.

[0490] (II) Verification of protein expression in the mouse CT26 tumor model using expression vector strains SWT5001, SWT5005, SWT6009, SWT6013 and control vector strains SWT7001, SWT7005, SWT7009, and SWT7013

[0491] This example was designed to demonstrate that the T7 expression vector can stably express the target protein in the in vivo tumor environment.

[0492] Tumor model mice were injected with the corresponding expression vectors and control vector strains through the tail vein, the tumor tissues were isolated, and the expression of the target protein was verified to detect the expression of the target protein of different Salmonella strains in the tumor model.

[0493] The specific steps are as follows:

[0494] 1. Tumor model establishment

[0495] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, raised in an SPF environment) were subcutaneously inoculated with 1×10 6 CT26 mouse colon cancer cells were used to establish a mouse colon cancer subcutaneous tumor model. 10-12 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into 8 groups (SWT5001, SWT5005, SWT6009, SWT6013, SWT7001, SWT7005, SWT7009, SWT7013), with 2 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0496] 2. Bacteria Preparation

[0497] Taking strain SWT1005 as an example, streak on an LB plate supplemented with D-alanine and culture statically in a 37°C constant temperature incubator overnight; pick strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 monoclonal clones and inoculate them 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, prepare a bacterial suspension in PBS, and administer it through the tail vein of mice.

[0498] 3. Tumor Extraction Sample Preparation

[0499] (1) On the third day after administration, mice were killed and tumor tissue was isolated. The tumor tissue was cut into 0.1 g tissue blocks using surgical scissors, ground using a homogenizer, and finally resuspended in 0.9 ml of 100 μg / ml D-alanine in fresh PBS solution. Lysis buffer (protein loading buffer, purchased from CST, product number 7722S) was added and resuspended. The solution was heated at 95°C for 10 minutes and centrifuged again at 12,000 rpm. The supernatant was recovered for later use.

[0500] (2) 5 μl of the supernatant recovered in the previous step was taken and added to SDS-PAGE gel (SDS-PAGE gel preparation kit purchased from Sangon Biotechnology, product number C631100-0200), and electrophoresed at 120 V for 60 minutes.

[0501] (3) The SDS-PAGE gel after electrophoresis was transferred to a PVDF membrane (purchased from Roche, product number 03010040001) at a steady current of 250 mA for 30 minutes.

[0502] (4) The transferred PVDF membrane was transferred to 5% skim milk powder and placed on a horizontal shaker at 80 rpm and incubated at room temperature for 1 hour as a blocking method.

[0503] (5) The blocked PVDF membrane was incubated with Anti-T7 RNA Polymerase antibody (T7 RNA polymerase antibody, purchased from Creative Biomart, catalog number CABT-B8990), Anti-diphtheria toxin antibody (DTA identification antibody, purchased from abcam, catalog number ab151222), and Anti-listeriolysin antibody (LLO identification antibody, purchased from abcam, catalog number ab200538) as primary antibodies diluted with 5% skim milk powder at 4°C for 18 hours (one antibody was used for each membrane).

[0504] (6) Dilute the secondary antibody with 5% skim milk powder, transfer the PVDF membrane after primary antibody incubation to the secondary antibody dilution solution, and incubate on a horizontal shaker at 80 rpm for 1 hour at room temperature.

[0505] (7) Wash the PVDF membrane after secondary antibody incubation with TBST Buffer (purchased from Sangon Biotechnology, product number C520009-0005) three times, 5 minutes each time. Add ECL developer (developer SignalFire) to the washed PVDF membrane. TM ECL Reagent (CST, Cat. No. 6883p3) was purchased and incubated in the dark for 1 min. After incubation, the PVDF membrane was placed on a chemiluminescence instrument for image acquisition.

[0506] In isolated tumor tissues, Western blot analysis revealed that strains SWT5001, SWT5005, SWT6009, and SWT6013 all expressed high levels of DTA (see Figure 23A ); LLO expression (see Figure 23B ); and T7 RNA polymerase expression (see Figure 23C ). However, in the control strains SWT7001, SWT7005, SWT7009, and SWT7013, only T7 RNA polymerase expression was detected (see Figure 23C ), with virtually no stable expression of DTA or LLO.

[0507] Example 10: Immunohistochemical analysis of tumor areas after injection of strains carrying expression vectors

[0508] To verify that Salmonella carrying the expression vector aggregates in the hypoxic area of ​​the tumor and expresses the target protein, this example uses SWT5005 as an example. The mouse tumor model injected with Salmonella through the tail vein was sectioned and stained to observe the distribution of the drug protein in the tumor model.

[0509] The specific steps are as follows:

[0510] 1. Tumor model establishment

[0511] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, raised in an SPF environment) were subcutaneously inoculated with 1×10 6 CT26 mouse colon cancer cells were used to establish a mouse colon cancer subcutaneous tumor model. 10-12 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS group and SWT5005 group, with 5 mice in each group. Tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0512] 2. Bacteria Preparation

[0513] Taking strain SWT5005 as an example, streak the plate onto an LB plate supplemented with 100 μg / ml D-alanine and culture it statically in a 37°C constant temperature incubator overnight. Pick a single colony of strain SWT5005 and inoculate it into 5 ml of fresh LB liquid medium containing 100 μg / ml D-alanine, and culture it in a constant temperature shaker at 37°C and 220 rpm for 16 hours. 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, prepare a bacterial suspension in PBS, and administer it to mice via the tail vein.

[0514] 3. Tumor sample slice analysis

[0515] On the 5th day after administration, mice were killed, tumor tissues were isolated and immersed in formalin fixative; gradient dehydration was performed, and the tissues were embedded in paraffin. After complete cooling and solidification, the sections were sliced ​​and dewaxed; the sections were immersed in sodium citrate antigen retrieval solution, boiled for 10 minutes for antigen retrieval, and then rinsed with PBS three times; the sections after antigen retrieval were immersed in 3% hydrogen peroxide solution for 10 minutes, and then rinsed with PBS three times; the sections 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-Salmonella (Salmonella identification marker, purchased from abcam, product number ab35156), anti-HIF1α (hypoxic zone identification marker, purchased from abcam, product number ab51608), anti-diphtheria Toxin (DTA identification antibody, purchased from abcam, catalog number ab151222) primary antibody incubation solution was added dropwise to different blocked tissue sections, incubated overnight at 4°C, and then rinsed three times with PBS; secondary antibody incubation solution was added dropwise to the tissue sections, incubated for 25 minutes, and then rinsed three times with PBS; DAB staining solution was added dropwise to the tissue sections, and counterstained with hematoxylin for 3 minutes; the tissue sections were sequentially dehydrated in a gradient manner, and after dehydration, the sections were mounted with mounting media, and the sections were observed under a microscope and photographed, as shown in Figure 24.

[0516] The results showed that after antibody staining comparison, the brown (dark) area was antibody-positive, and Salmonella containing the hypoxia-specific gene expression cassette was found to be aggregated in the hypoxic area of ​​the tumor, as shown by the arrows in Figures 24A and 24B , and the expressed drug DTA was found to be widely distributed in the tumor, as shown by the arrow in Figure 24C .

[0517] Example 11: Verification of the killing effect of strains carrying expression vectors and control vectors on cancer cells under hypoxic conditions

[0518] The cells were co-cultured with cancer cells under hypoxic conditions in vitro to verify the killing effects of strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, and SWT7013 on various cancer cells.

[0519] The specific steps are as follows:

[0520] 1. Streak strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 on LB plates containing 100 μg / ml D-alanine and culture them statically in a 37°C incubator overnight.

[0521] 2. Pick single clones of strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 and inoculate them 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.

[0522] 3. Measure the absorbance of the strain at 600 nm, take the culture with an OD value of 1, wash it twice with PBS, then wash it once with the corresponding cell culture medium, and then resuspend it in the cell culture medium (infection ratio is 200:1).

[0523] 4. The above bacteria were co-cultured with 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) at an oxygen concentration below 0.8% for 2 hours. The cells were then washed three times with PBS containing gentamicin, and the medium was changed and cultured in an anaerobic jar at an oxygen concentration below 0.8% for 24 hours. The cells were photographed and recorded using a 20x objective lens under white light, as shown in Figures 25-29.

[0524] 5. Mix CCK8 reagent with the above cell suspension and incubate for 1 hour.

[0525] 6. Use an enzyme-labeled instrument to measure the absorbance (detection wavelength 450 nm) and calculate the percentage of cytotoxicity.

[0526] The killing effects of strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 on EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7 and MB49 cells are shown in Figures 30 and 31.

[0527] The experimental results were analyzed using T-test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0528] The above results show that the Salmonella strains SWT5001, SWT5005, SWT6009, and SWT6013 carrying the expression vector all have obvious killing effects on various cancer cells. Significant cell death can be found under a microscope under white light, and analysis of the death reagent CCK8 found that their cell killing effect is stronger than that of the control strains SWT7001, SWT7005, SWT7009, and SWT7013, indicating that Salmonella carrying the three-in-one drug expression vector of the T7 expression system has universal killing effects on cancer cells.

[0529] Example 12: Evaluation of tumor suppressive properties of strains carrying expression vectors and control vectors in mouse tumor models

[0530] To verify the inhibitory effect of strains carrying expression vectors on mouse tumor growth, the present invention designed SWT5005 and SWT7005 as representatives to conduct inhibitory experiments in mouse EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7 and MB49 tumor models.

[0531] 1. Establishment of various mouse tumor models

[0532] (1) Establishment of EMT6 tumor mouse model

[0533] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 EMT6 cancer cells were used to establish a mouse breast subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0534] (2) Establishment of K7M2 tumor mouse model

[0535] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 K7M2 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0536] (3) Establishment of Hepa1-6 tumor mouse model

[0537] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Hepa1-6 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0538] (4) Establishment of A549 tumor mouse model

[0539] Nu / Nu nude mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 A549 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0540] (5) Establishment of B16F10 tumor mouse model

[0541] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 B16F10 cancer cells were used to establish a mouse breast subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 100 mm 3The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0542] (6) Establishment of Renca tumor mouse model

[0543] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Renca cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0544] (7) Establishment of MFC tumor mouse model

[0545] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 MFC cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0546] (8) Establishment of Pan02 tumor mouse model

[0547] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Pan02 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×107 CFU bacteria.

[0548] (9) Establishment of RM-1 tumor mouse model

[0549] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 RM-1 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0550] (10) Establishment of CT26 tumor mouse model

[0551] BALB / c mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 CT26 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0552] (11) Establishment of ID8 tumor mouse model

[0553] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 ID8 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The experiment was carried out at about 125 μl. The mice were divided into 3 groups: PBS control group, SWT5005 and SWT7005, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0554] (12) Establishment of Neuro-2a tumor mouse model

[0555] A / J mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Neuro-2a cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0556] (13) Establishment of SCC7 tumor mouse model

[0557] Nu / Nu nude mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 SCC7 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0558] (14) Establishment of MB49 tumor mouse model

[0559] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and housed in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 MB49 cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100 mm 3 The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated into the tail vein with 1×10 7 CFU bacteria.

[0560] 2. Bacterial preparation and treatment

[0561] The constructed Salmonella strains SWT5005 and SWT7005 were selected and inoculated into 5 ml of LB liquid medium supplemented with D-alanine, and cultured in a constant temperature shaker at 37°C and 220 rpm for 16 hours. The absorbance of the strains at 600 nm was measured, and the culture with an OD value of 1 was measured and washed three times with PBS. 1 × 10 7 CFU count bacteria were administered to model mice; the day of administration was designated as day 0, and the length and width of the mouse tumors were measured on days 1, 3, 5, 7, 9, and 11 of administration, and the mouse tumor volume was calculated according to the following formula: Mouse tumor volume = length × width 2 ×0.52

[0562] The tumor inhibitory effects of SWT5005 and SWT7005 in mouse tumor models are shown in Figures 32-35 .

[0563] The experimental results were analyzed using T-test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0564] The results showed that the Salmonella strain SWT5005 carrying the expression vector had a significant inhibitory effect on tumor growth, which was better than the control vector strain SWT7005 and the untreated control.

[0565] The information of the corresponding strains of the present invention is shown in Table 2.

[0566] Table 2 Strain construction table of the present invention

[0567] The information of the corresponding tool plasmids of the present invention is shown in Table 3.

[0568] Table 3 Information of tool plasmids used in the present invention

[0569] The sequences of promoter, gene coding region, protein, etc. used in the present invention are shown in Table 4.

[0570] Table 4 Specific sequences of promoters, gene coding regions, proteins, etc. used in the present invention

[0571] The specific sequences of all primers used in the present invention are shown in Table 5.

[0572] Table 5 Specific sequences of all primers used in the present invention

[0573] The present application has been described above in conjunction with preferred embodiments, but these embodiments are merely exemplary and serve only as an illustrative example. On this basis, various replacements and improvements can be made to the present application, all of which fall within the scope of protection of the present application.

Claims

1. A constitutive plasmid expression vector, which contains two phage-derived promoters, which respectively control the expression of a drug protein gene and a membrane-breaking protein gene, and the phage-derived promoter binds to an RNA polymerase encoded by a specific phage RNA polymerase gene on the chromosome of a host bacterium.

2. The expression vector according to claim 1, wherein the dual phage-derived promoter is selected from the group consisting of T7, T3 and SP6 promoters.

3. The expression vector according to claim 2, wherein the biphage-derived promoter is a T7 promoter.

4. The expression vector according to any one of claims 1 to 3, wherein the promoters derived from the two phages are oriented in opposite directions, thus forming an opposing promoter expression vector.

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

6. An expression vector according to any preceding claim, comprising a replicon essential for plasmid replication.

7. The expression vector according to claim 6, wherein the replicon is selected from the group consisting of pUC, p15A, ColE1 and R6K.

8. The expression vector according to claim 7, wherein the replicon is pUC.

9. The expression vector according to any one of the preceding claims, wherein the drug protein gene is the A fragment of the AB type exotoxin drug gene of bacterial origin.

10. The expression vector according to claim 9, wherein the drug protein gene is selected from the group consisting of diphtheria toxin A fragment gene, cholera toxin A fragment gene, Shiga toxin A fragment gene, pertussis toxin A fragment gene and Pseudomonas exotoxin A fragment gene.

11. The expression vector according to claim 10, wherein the drug protein gene is a diphtheria toxin A fragment gene.

12. The expression vector according to any one of the preceding claims, wherein the membrane permeabilin gene is selected from the listeriolysin hlyA gene.

13. An expression vector according to any preceding claim, wherein the host bacterium is a Gram-negative bacterium.

14. A method for constructing the expression vector according to any one of the above claims, comprising the following steps: (1) sequentially connecting the phage-derived promoter, ribosome binding site and membrane-breaking protein gene described in any one of the above claims to construct a membrane-breaking protein gene expression unit; (2) sequentially connecting the bacteriophage-derived promoter, ribosome binding site and drug protein gene described in any of the above claims to construct a drug protein gene expression unit; (3) Connect the plasmid replicon, asd gene, and the above two gene expression units together to form a complete plasmid expression vector.

15. A modified Gram-negative bacterium comprising the expression vector according to any one of the above claims. The Gram-negative bacterium according to claim 15 , wherein the specific bacteriophage RNA polymerase gene is a gene corresponding to a promoter derived from a biphage.

17. The Gram-negative bacterium according to claim 16, wherein the specific bacteriophage RNA polymerase gene is selected from the group consisting of a T7 RNA polymerase gene, a T3 RNA polymerase gene and a SP6 RNA polymerase gene. The Gram-negative bacterium according to claim 17 , wherein the specific bacteriophage RNA polymerase gene is a T7 RNA polymerase gene.

19. The Gram-negative bacterium according to any one of claims 15 to 18, wherein the expression of the specific bacteriophage RNA polymerase gene is regulated by a constitutive expression promoter.

20. The Gram-negative bacterium according to claim 19, wherein the constitutive expression promoter is a lacUV5 promoter.

21. The Gram-negative bacterium according to any one of claims 15 to 20, further comprising a hypoxia-specific gene expression cassette, wherein the hypoxia-specific gene expression cassette comprises: a) a positive hypoxia promoter, which is a promoter comprising an FNR binding site, and the positive hypoxia promoter can be induced to express under hypoxia; b) genes essential for survival; and c) 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.

22. The Gram-negative bacterium according to claim 21, 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.

23. The Gram-negative bacterium according to claim 21 or 22, wherein the forward hypoxia promoter and / or the reverse hyperoxia promoter of the hypoxia-specific gene expression cassette are promoters derived from Gram-negative bacteria.

24. The Gram-negative bacteria according to any one of claims 21 to 23, wherein The positive hypoxia promoter is selected from the promoter region sequence of yhbU or ynfK; Genes essential for survival are selected from alr or dadX; The inverted hyperoxic promoter is selected from the promoter region sequence of cyoA or ydcI.

25. The Gram-negative bacterium of claim 24, wherein 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.

26. The Gram-negative bacterium according to claim 24, wherein the positive hypoxic promoter is ynfK, which is selected from the group consisting of Salmonella, Escherichia coli, Serratia, Shigella and Enterobacter reuteri.

27. The Gram-negative bacterium according to claim 24, wherein the alr gene and the dadX gene are selected from the group consisting of Salmonella, Escherichia coli, Shigella, Klebsiella, Yersinia, Haemophilus and Pseudomonas.

28. The Gram-negative bacterium according to claim 24, wherein the hypoxia-specific gene expression cassette consists of a forward hypoxia promoter yhbU, a survival essential gene alr, and a reverse hyperoxia promoter cyoA.

29. The Gram-negative bacterium according to claim 24, wherein the hypoxia-specific gene expression cassette consists of a forward hypoxia promoter yhbU, a survival essential gene alr, and a reverse hyperoxia promoter ydcI.

30. The Gram-negative bacterium according to claim 24, wherein the hypoxia-specific gene expression cassette consists of a positive hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperoxia promoter cyoA.

31. The Gram-negative bacterium according to claim 24, wherein the hypoxia-specific gene expression cassette consists of a positive hypoxia promoter ynfK, a survival essential gene dadX, and a reverse hyperoxia promoter ydcI.

32. The Gram-negative bacterium according to any one of claims 21 to 31, wherein the hypoxia-specific gene expression cassette is regulated by oxygen concentration.

33. The Gram-negative bacterium according to claim 32, wherein the forward hypoxic promoter functions when the oxygen content is below 1%, but fails to function when the oxygen content is above 1%; and / or the reverse hyperoxic promoter functions when the oxygen content is above 1%, but fails to function when the oxygen content is below 1%.

34. The Gram-negative bacterium according to claim 33, wherein the forward hypoxic promoter functions when the oxygen content is lower than 0.8%, but fails to function when the oxygen content is higher than 0.8%; and / or the reverse hyperoxic promoter functions when the oxygen content is higher than 0.8%, but fails to function when the oxygen content is lower than 0.8%.

35. A method for controlling the expression of a drug protein in a prokaryotic cell using the expression vector according to any one of claims 1 to 13, comprising the following steps: (1) preparing the expression vector according to any one of claims 1 to 13; (2) integrating the constitutively expressed bacteriophage RNA polymerase gene expression cassette into the host bacterial chromosome; (3) Knock out the asd gene on the host bacterial chromosome; (4) Transforming the expression vector according to any one of the above claims into a host bacterium.

36. The method of claim 35, further comprising integrating the hypoxia-specific gene expression cassette of any one of claims 21-34 into a host bacterial chromosome.

37. A method according to claim 35 or 36, wherein the host bacterium is a Gram-negative bacterium.

38. The Gram-negative bacteria according to any one of the above claims, 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, Vibrio parahaemolyticus, Vibrio cholerae, Yersinia pestis, Catarrhalis, Moraxella catarrhalis, Campylobacter jejuni, Shigella dysenteriae, Neisseria gonorrhoeae, Haemophilus influenzae, Moraxella, Neisseria meningitidis, Proteus vulgaris, Proteus mirabilis, Pasteurella haemolyticus, Legionella pneumophila, Yersinia pestis, Shigella sonnei, Pseudomonas aeruginosa, Yersinia enterocolitica, Cryptococcus neoformans, Burkholderia cepacia, and Helicobacter pylori.

39. The Gram-negative bacterium according to claim 38, which is Salmonella.

40. The Gram-negative bacterium according to claim 38 or 39, which is an attenuated Gram-negative bacterium.

41. The Gram-negative bacterium of claim 40, wherein the attenuated Gram-negative bacterium is Salmonella.

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

43. The pharmaceutical composition according to claim 42, further comprising a pharmaceutically acceptable carrier.

44. The pharmaceutical composition of claim 43, wherein the pharmaceutically acceptable carrier is selected from the group consisting of disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants and lubricants.

45. A pharmaceutical composition according to any one of claims 42-44, for use in the treatment of solid tumors.

46. ​​Use of the expression vector according to any one of the above claims, the Gram-negative bacteria according to any one of the above claims, or the pharmaceutical composition according to any one of the above claims in the preparation of tumor drugs.

47. The use according to claim 46, wherein the tumor is a solid tumor.

48. The pharmaceutical composition of claim 45 or the use of claim 47, wherein the solid tumor is selected from the group consisting of breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal (colon cancer, rectal cancer), pelvic (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer, and bladder tumors / cancers.

49. The pharmaceutical composition or use according to claim 48, wherein the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma and bladder cancer.

50. A method for treating a tumor, comprising administering to a subject the expression vector of any one of claims 1-13, the Gram-negative bacteria of any one of claims 15-34 and 38-41, or the pharmaceutical composition of any one of claims 42-45.

51. The method of claim 50, wherein the tumor is a solid tumor.

52. The method of claim 51, wherein the solid tumor is selected from the group consisting of tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestinal (colon cancer, rectal cancer), pelvic (cervical cancer, ovarian malignancies, endometrial cancer, ovarian cancer), nervous system, head and neck cancer, and bladder.

53. The method of claim 52, wherein the solid tumor is breast cancer, osteosarcoma, liver cancer, lung cancer, melanoma, kidney cancer, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, ovarian cancer, neuroblastoma, squamous cell carcinoma, and bladder cancer.

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