Constitutive expression vector containing double-phage-source promoter, bacteria containing same and application of constitutive expression vector

Abstract

The invention belongs to the field of genetic engineering and biological treatment, and provides a constitutive expression vector containing a double-phage-source promoter, gram-negative bacteria containing the expression vector and application of the gram-negative bacteria in preparation of tumor drugs. An expression vector is divided into a protein expression control module, a drug delivery module and a drug killing module by selecting a promoter from bacteriophage, a specific bacteriophage RNA polymerase gene, a membrane rupture protein gene and a drug protein gene. Through organic combination of the three modules, a three-in-one system is formed, and the system and method for stably and efficiently carrying out drug protein expression, drug delivery and drug killing are realized.

Description

Technical Field

[0001] The invention relates to a constitutive expression vector containing a double phage-derived promoter, bacteria containing the same and application thereof, and belongs to the field of genetic engineering and biological treatment. Background Art

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

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

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

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

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

[0007] Studies have shown that after 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.

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

[0009] First, the intensity and stability of target drug expression.

[0010] The main factor considered in previous bacterial in vitro expression technology is 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 the in vivo tumor environment, the bacteria themselves will face the pressure of the complex tumor microenvironment (including hypoxia, low pH, high lactic acid, immune cells against bacteria and other extreme environments), 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 of drug proteins will also be affected, thereby affecting the effect of tumor inhibition.

[0011] Second, the choice of medicine is very important.

[0012] The environment inside a tumor is not completely closed, so if anti-tumor drugs are released directly inside the tumor, some of the drugs will diffuse along the blood vessels to other organs and tissues, causing unnecessary side effects. Therefore, it is particularly important to choose a drug that can only kill tumors inside tumor cells.

[0013] Third, how the drug is delivered is very important.

[0014] If drugs are to be delivered into tumor cells, bacteria need to invade tumor cells first and then release drugs inside the tumor cells. Since bacteria invade cells by inducing endocytosis of tumor cells to form phagocytic vesicles, drugs synthesized by bacteria need to pass through vesicles to reach the cytoplasm of tumor cells to exert their effects, and the release efficiency of natural bacteria is very low. Therefore, a highly efficient mode of penetrating vesicles to release drugs is needed to increase the efficiency of drug release.

[0015] 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 in tumors in vivo. Summary of the invention

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

[0017] In view of 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.

[0018] To this end, the present invention provides a constitutive plasmid expression vector, which contains promoters from two phage sources, which respectively control the expression of drug protein genes and membrane-breaking protein genes, and integrates a specific phage RNA polymerase gene controlled by a constitutively expressed promoter on the host bacterial chromosome.

[0019] 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.

[0020] Through the organic combination of the above three modules and 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.

[0021] (1) Protein expression control module

[0022] 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.

[0023] The present invention preferably uses the constitutive artificial promoter lacUV5 to control the expression of the phage-derived T7 RNA polymerase gene, and integrates this expression cassette into the chromosome of the bacterium to achieve stable inheritance. Since the T7 RNA polymerase specifically recognizes the T7 promoter, the gene controlled by the T7 promoter can be stably and highly expressed. Therefore, the present invention selects to place the drug delivery module gene and the drug killing module gene behind the T7 promoter respectively, and places them in a plasmid expression vector for expression regulation.

[0024] (2) Drug delivery module

[0025] When bacteria invade tumor cells, phagocytic vesicles are formed. The presence of phagocytic vesicles will limit the further release of bacteria and the drugs synthesized by them into the cells. In order to solve this problem, the present invention preferably uses listeriolysin-O (LLO) as a membrane-breaking protein to achieve the purpose of drug delivery.

[0026] (3) Drug killing module

[0027] In order to achieve precise drug delivery that only exerts a killing effect in tumor cells, the present invention uses the A fragment of the bacterial AB type exotoxin protein to achieve precise intracellular killing. Because the characteristic of bacterial AB type exotoxin protein is that the exotoxin protein can be divided into two independent functional regions, among which 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.

[0028] 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, which promotes 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 cells, 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.

[0029] It can be seen that the present invention realizes 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.

[0030] 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.

[0031] 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).

[0032] In a more preferred embodiment of the present invention, the dual phage-derived promoter is T7 promoter (SEQ ID No. 1).

[0033] In addition, according to the different directions of the promoters from the two phages, the directions of the promoters from the two phages in the preferred expression vector of the present invention are opposite, that is, an opposing promoter expression vector, as shown in the attached Fig.18 shown.

[0034] In addition, in order 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 Salmonella loses the plasmid vector it carries, the bacteria will die quickly due to the lack of essential genes, and the surviving bacteria will all carry the plasmid expression vector, thus ensuring that the entire system can stably and efficiently express protein drugs.

[0035] The asd gene (SEQ ID No. 11) and the protein sequence of asd (SEQ ID No. 12) of the genus Salmonella encode aspartate B-semialdehyde dehydrogenase, which is an enzyme required in the process of synthesizing diaminopimelic acid (DAP), an important component of the cell wall of Gram-negative bacteria. Knocking out the asd gene of Salmonella will cause the lysis and death of Salmonella. However, when DAP is supplemented in the culture medium or by making Salmonella carry a vector containing the asd gene, the normal growth of Salmonella can be maintained.

[0036] 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.

[0037] 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.

[0038] Meanwhile, the expression vector of the present invention does not contain a resistance gene.

[0039] In a preferred embodiment of the present invention, the replicon is selected from the group consisting of pUC, p15A, ColE1 and R6K.

[0040] In a more preferred embodiment of the present invention, the replicon is pUC (SEQ ID No. 36).

[0041] In order to stably and efficiently carry out drug killing, the present invention selects drug proteins with high efficiency and specific killing power, which can prevent accidental damage to other organs after drug protein leakage while killing tumor cells.

[0042] 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.

[0043] In a preferred embodiment of the present invention, the drug 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.

[0044] Diphtheria toxin (DT) is a protein toxin composed of two relatively independent subunits (A and B) connected by disulfide bonds.

[0045] The A subunit (DTA) has enzymatic activity and can inactivate elongation factor-2 (EF-2) by catalyzing the glycosylation of ADP, thereby blocking the synthesis of eukaryotic cell proteins. DTA is the main source of diphtheria toxin cytotoxicity.

[0046] The B subunit (DTB) is non-cytotoxic and can bind to the surface receptors of eukaryotic cells, thereby mediating the entry of the A subunit into the cells. Based on the independent functions of diphtheria toxin DTA and DTB, when DTA is independently expressed in bacteria but does not enter eukaryotic cells, it will not cause harm to bacteria and eukaryotic cells. Only when the DTA protein is released into eukaryotic cells will it be toxic to eukaryotic cells.

[0047] 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 of A subunit and B subunit. For example, cholera toxin A subunit fragment gene (SEQ ID No.17) and its protein sequence (SEQ ID No.18), Shiga toxin gene Stx1 A fragment (SEQ ID No.19) and protein sequence (SEQ ID No.20), Shiga toxin gene Stx2 A fragment (SEQ ID No.21) and protein sequence (SEQ ID No.22), pertussis toxin A subunit fragment gene (SEQ ID No.23) and protein sequence (SEQ ID No.24), Pseudomonas exotoxin A subunit fragment gene (SEQ ID No.25) and protein sequence (SEQ ID No.26).

[0048] Cholera toxin (CT) originates from Vibrio cholerae and is composed of two subunits, A and B. Subunit A is an ADP-ribosyltransferase that causes cell dehydration and death by disrupting G protein signal transduction. Cholera toxin subunit B (CTB) is attached to the cell surface by binding to the pentose chain of ganglioside GM1 and can be absorbed by the axon terminal and transported back to the cell body.

[0049] The Shiga toxin gene is a family of related toxins with two major classes: Stx1 and Stx2. The toxin has two subunits, A and B, which are one of the AB toxins. The B subunit is a pentamer that binds to specific glycolipids on the host cell, specifically triacylceramide (Gb3). Binding of the B subunit to Gb3 causes the induction of narrow tubular membrane invaginations, driving the formation of endomembrane tubules for bacterial uptake into the cell. Once inside the cell, the A subunit cleaves specific adenine nucleobases from the 28S RNA of the ribosomal 60S subunit, thereby stopping protein synthesis.

[0050] 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 transferred to host cells after a conformational change in the membrane-bound transporter B subunit. Pertussis toxin is an exotoxin with six subunits (named S1 to S5, each complex contains two copies of S4). The subunits are arranged in an AB structure: the A component is enzymatically active and is formed by the S1 subunit, while the B component is the receptor-binding part and consists of subunits S2-S5. PT is released from Bordetella pertussis in an inactive form. After binding to the cell membrane receptor, PT is taken up by the endosome and then retrogradely transported to the trans-Golgi network and the endoplasmic reticulum. During the transport process, the A subunit is activated by glutathione and ATP to catalyze the ADP-ribosylation of the heterotrimeric G protein, thereby failing to inhibit adenylate cyclase activity, resulting in an increase in intracellular cAMP concentration and cell death.

[0051] Pseudomonas exotoxin is an AB-type exotoxin produced by Pseudomonas aeruginosa. The A subunit converts NAD + The toxin transfers the 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).

[0052] In order to deliver drugs stably and efficiently, the present invention designs a precise drug delivery system, which aims to accurately release the drug protein synthesized by bacteria into tumor cells, which is further divided into two steps: bacteria invading host cancer cells and releasing synthesized drug proteins.

[0053] 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.

[0054] Salmonella enterica is able to invade and replicate in host cells, including epithelial cells and macrophages. This ability is present in the pathogenicity island 1 of Salmonella. Through the type III secretion system 1 (T3SS1), Salmonella secretes T3SS effector proteins, which induce a large amount of actin rearrangement in the host cell, leading to membrane ruffles, thereby forming phagocytic vesicles containing Salmonella. The survival and proliferation of Salmonella in the phagocytic vesicles are then promoted by the type III secretion system 2 (T3SS2) encoded by the Salmonella pathogenicity island 2 (SPI-2) gene.

[0055] 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 itself and optimized by Salmonella codons.

[0056] 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.

[0057] LLO has a pH-sensitive acidic domain. The optimal pH for LLO precursor protein to mature and begin to exert membrane perforation activity is about 5.0-5.5, while it is inactive at neutral pH.

[0058] When Salmonella invades and forms phagocytic vesicles, the phagocytic vesicles will fuse with intracellular vacuoles such as lysosomes, thereby greatly reducing the pH. Under this condition, the activity of LLO is activated, playing a perforating role, allowing Salmonella to release the synthesized drug protein.

[0059] Therefore, in a preferred embodiment of the present invention, the membrane permeabilin gene is the listeriolysin hlyA gene.

[0060] In a preferred embodiment of the present invention, in the double phage-derived promoter, one of them is sequentially connected to the ribosome binding site (Ribosomal Binding Site, RBS) (SEQ ID No.35) and the membrane-permeabilizing protein gene, and the other is sequentially connected to the ribosome binding site (Ribosomal Binding Site, RBS) (SEQ ID No.35) and the drug protein gene.

[0061] Furthermore, in a preferred embodiment of the present invention, the host bacteria of the expression vector are Gram-negative bacteria.

[0062] Another aspect of the present invention provides a method for constructing the plasmid expression vector of the present invention, comprising the following steps:

[0063] 1. Sequentially connect the phage-derived promoter, ribosome binding site and membrane-breaking protein gene described in the present invention to construct a membrane-breaking protein gene expression unit;

[0064] 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;

[0065] 3. Connect the plasmid replicon, asd gene, and the above two gene expression units together to form a complete plasmid expression vector.

[0066] Another aspect of the present invention provides a modified Gram-negative bacterium comprising the expression vector of the present invention.

[0067] In a preferred embodiment of the present invention, the specific bacteriophage RNA polymerase gene is a gene corresponding to a promoter derived from a biphage.

[0068] In a more preferred embodiment of the present invention, the specific bacteriophage 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).

[0069] In a further preferred embodiment of the present invention, the specific bacteriophage RNA polymerase gene is a T7 RNA polymerase gene.

[0070] 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.

[0071] In order to achieve higher intensity expression, in a preferred embodiment of the present invention, an RNA polymerase expression system of T7 bacteriophage is used, which includes T7 RNA polymerase and T7 promoter to control the expression of the target drug protein.

[0072] The T7 RNA polymerase system is derived from the expression system of E. coli T7 phage. It is widely used in E. coli gene expression due to its strong protein expression ability. The characteristic of T7 RNA polymerase is that it can specifically recognize the T7 promoter without being affected by environmental factors. At the same time, the RNA synthesis rate of T7 RNA polymerase is five times that of E. coli RNA polymerase, so it can express the target protein with high intensity.

[0073] In a preferred embodiment of the present invention, T7 polymerase and T7 promoter can be replaced by T3 polymerase and T3 promoter. Similar to the T7 RNA polymerase system, the T3 RNA polymerase system is a highly specific RNA polymerase system derived from T3 bacteriophage that recognizes the T3 promoter sequence. Alternatively, T7 polymerase and T7 promoter can be replaced by SP6 polymerase and SP6 promoter. Similar to the T7 RNA polymerase system, SP6 polymerase is also a highly specific RNA polymerase system derived from SP6 bacteriophage that 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.

[0074] In order to achieve stable expression without being affected by bacterial internal metabolism and the microenvironment within the tumor, in a preferred embodiment of the present invention, the expression of the specific bacteriophage RNA polymerase gene is regulated by a constitutive expression promoter.

[0075] 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.

[0076] The lacUV5 promoter is very similar to the classic lac promoter. It contains only 2 base pair mutations in the -10 region compared to the lac promoter. The LacUV5 promoter does not require an additional activator and can drive high levels of gene expression. Although no activator is required, expression of the lacUV5 promoter is regulated by the LacI repressor in E. coli and can be induced by IPTG. IPTG is an effective inducer when used in a concentration range of 100 μM to 1.5 mM.

[0077] Since Salmonella has lost the lacI gene and the entire lac operon during evolution, the use of the lacUV5 promoter in Salmonella is an excellent constitutive expression system.

[0078] 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.

[0079] In a preferred embodiment of the present invention, the Gram-negative bacteria further comprises a hypoxia-specific gene expression cassette comprising:

[0080] 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;

[0081] b) genes essential for survival; and

[0082] 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;

[0083] The survival essential gene is a gene encoding alanine racemase.

[0084] "Polypeptide", "peptide", and "protein" are used interchangeably in the present invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acids, as well as to polymers of natural amino acids. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms, including but not limited to glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

[0085] In the present invention, "polynucleotide" refers to a macromolecule formed by connecting multiple nucleotides through phosphodiester bonds, wherein the nucleotides include ribonucleotides and deoxyribonucleotides. The sequence of the polynucleotide of the present invention can be codon-optimized for different host cells (such as Escherichia coli) to improve the expression of the polypeptide. Methods for codon optimization are known in the art.

[0086] In order for bacteria to survive only in low oxygen concentration conditions, three requirements need to be met:

[0087] 1) Control the strength of the upstream promoter and the background leakage expression.

[0088] If the oxygen sensor partially leaks expression without binding to the upstream promoter, it will make it impossible to regulate oxygen, resulting in the bacteria surviving at any oxygen concentration; or if the strength of the upstream promoter is too weak, it will not be able to start the expression of the downstream gene, resulting in the bacteria not surviving at any oxygen concentration;

[0089] 2) Select appropriate genes essential for survival.

[0090] By selecting genes essential for survival, it can be ensured that the bacteria die when the genes essential for survival are not expressed, and the survival of the bacteria can be quickly ensured when the expression is induced by hypoxia;

[0091] 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.

[0092] Therefore, in the present invention, "hypoxia-specific gene expression cassette" refers to a piece of DNA that can initiate expression of essential genes under hypoxic conditions, which contains an essential gene controlled by a hypoxia-inducible promoter and, if necessary, may further contain other regulatory elements required for the expression of the essential gene.

[0093] In the present invention, "essential gene" refers to a gene that plays a decisive role in the growth and / or survival of bacteria. Once the bacteria lack this gene or its functional expression product, they cannot survive, divide and / or grow normally. A typical example of lacking an essential gene or its functional expression product is a nutrient-deficient strain, which cannot survive, divide and / or grow normally under in vitro culture conditions or in vivo environments in the absence of specific exogenous supplements. Essential genes usually exist in a single copy on bacterial chromosomes.

[0094] From this, we can see that the requirements for genes essential for survival are as follows:

[0095] (1) It is an essential gene for bacterial reproduction. If it is missing, it will lead to rapid death of bacteria;

[0096] (2) The product of this gene does not exist in the normal environment or the human body, which ensures that it will not get out of control in the human environment, and can facilitate the cultivation and preparation of bacteria in a normal culture environment by adding the corresponding expression product of this gene;

[0097] (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.

[0098] In the present invention, the survival essential gene is a gene encoding alanine racemase.

[0099] For Gram-negative bacteria such as Escherichia coli and Salmonella, the cell wall is an indispensable component. The core component of the cell wall is peptidoglycan. When synthesizing peptidoglycan, bacteria need D-alanine as an important component. If D-alanine is missing, the bacteria will not be able to synthesize the cell wall and will then lyse.

[0100] All amino acids that exist in nature are L-type, so there are two genes in Gram-negative bacteria: the alr gene for the biosynthesis of alanine racemase and dadX, which are responsible for converting L-alanine into D-alanine to meet the needs of cell wall synthesis.

[0101] 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.

[0102] In our previous research, YB1 Salmonella used asd gene as essential gene for regulation. Compared with YB1 Salmonella, the present invention selected alr gene and dadX gene as essential genes for survival. alr gene and dadX gene are functionally homologous genes. Therefore, in the present invention, the gene of alanine racemase can be alr gene (SEQ ID No.27) from Salmonella and protein sequence of alr of Salmonella (SEQ ID No.28) or dadX gene (SEQ ID No.29) from Salmonella and protein sequence of dadX of Salmonella (SEQ ID No.30); or alr or dadX gene of other Gram-negative bacteria or genes with equivalent functions.

[0103] In the present invention, alr gene knockout bacteria are first constructed, and then another gene dadX is modified, so that the bacteria become alr gene and dadX gene defective bacteria after editing, and a forward hypoxia promoter and a reverse hyperoxia promoter are used to regulate the additional alr or dadX gene.

[0104] The positive hypoxia promoter of the present invention is a hypoxic promoter regulated by FNR; the reverse hyperoxic promoter of the present invention is an antisense promoter negatively regulated by FNR and ArcA. The fumarate and nitrate reduction gene fnr is an important gene regulating the aerobic and anaerobic growth of Salmonella. This complex regulatory system has been widely studied in Escherichia coli and Salmonella. The DNA binding protein FNR encoded by the fnr gene senses changes in oxygen and controls the expression of different genes, achieving switching at the overall metabolic level. Therefore, DNA binding sequences such as FNR and ArcA become an important way to control the expression of downstream genes.

[0105] FNR has an oxygen-sensitive [4Fe-4S] 2+ The domain can directly sense oxygen and regulate site-specific DNA binding. In contrast, ArcA senses signals from the aerobic respiratory chain. Therefore, there are two different mechanisms for sensing changes in oxygen concentration in Gram-negative facultative anaerobes such as Salmonella and Escherichia coli. When FNR regulates gene expression in hypoxia, it is divided into two situations: activation and inhibition.

[0106] 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.

[0107] Among them, the positive hypoxia promoter used in the present invention is as follows:

[0108] (1) Salmonella yhbU promoter (yhbU-S) (SEQ ID No. 31), wherein the FNR binding site is "CTGCCTTAAATCAA";

[0109] (2) Escherichia coli ynfK promoter (ynfK-E) (SEQ ID No. 32), wherein the FNR binding site is "TTGCGCTATCTCAA";

[0110] Among them, the reverse high oxygen promoter used in the present invention is as follows:

[0111] (1) Salmonella cyoA promoter (cyoA-S) (SEQ ID No. 33), wherein the binding site of FNR is "TTTATTGATAATAA" and the binding site of ArcA is "GTTAAGTA";

[0112] (2) Salmonella ydcI promoter (ydcI-S) (SEQ ID No. 34), wherein the binding site of FNR is "GTTATCAAAAACAA" and the binding site of ArcA is "GTTAATAA";

[0113] Through the analysis of the above-mentioned reverse hypoxia promoter and forward hyperoxia promoter, we found that the FNR binding sites all conform to the "TTGATNNNNATCAA" pattern, and any base in the TTGAT and ATCAA sequences in the conserved binding sites can be replaced, but the total number does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; and ArcA conforms to the "GTTAATTA" pattern of its core region, and any base in the GTTAATTA sequence in the conserved binding site can be replaced, but the total number does not exceed 2.

[0114] Therefore, in a preferred embodiment of the present invention, the FNR binding site of the forward hypoxia promoter or the reverse hyperoxia promoter conforms to the pattern of TTGATNNNNATCAA, N is any base of A, T, C, G, and any base in the TTGAT and ATCAA sequences in the conserved binding site can be replaced, but the total number of replacements does not exceed 3, and 3 consecutive adjacent bases cannot be replaced; the ArcA binding site of the reverse hyperoxia promoter conforms to the pattern of GTTAATTA, wherein any base can be replaced, but the total number of replacements does not exceed 2.

[0115] In a preferred embodiment of the present invention, the forward hypoxia promoter and / or the reverse hyperoxia promoter of the hypoxia-specific gene expression cassette is a promoter derived from Gram-negative bacteria.

[0116] 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.

[0117] In a further preferred embodiment of the present invention, the positive hypoxic promoter is yhbU, which is selected from the group consisting of Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia and Simhuisella.

[0118] 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.

[0119] 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.

[0120] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene alr and a negative hyperoxia promoter cyoA.

[0121] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter yhbU, a survival essential gene alr and a negative hyperoxia promoter ydcI.

[0122] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter ynfK, a survival essential gene dadX and a negative hyperoxia promoter cyoA.

[0123] In a further preferred embodiment of the present invention, the expression cassette consists of a positive hypoxia promoter ynfK, a survival essential gene dadX and a negative hyperoxia promoter ydcI.

[0124] The positive hypoxia promoter disclosed in patent CN104471057B is pepT. The pepT promoter is not completely regulated by FNR, but is a promoter that is dual-regulated by CRP-cAMP and FNR. Therefore, half of the FNR binding site in the pepT promoter is a binding site for CRP, and half is a binding site for FNR. The CRP-FNR binding region sequence of the pepT promoter is GTGACCTGACGCAA, of which the first half GTGA conforms to the first half of the CRP conservative binding site GTGANNNNNNTCAC, and the second half CGCAA conforms to the part of the FNR conservative region ATCAA, the tenth A is replaced by C, and the eleventh T is replaced by G, which does not conform to the rules for the positive hypoxia promoter in the present invention.

[0125] The FNR binding region TTGATAATCATTTT of the reverse hyperoxia promoter sodA disclosed in patent CN104471057B only contains the first half region TTGAT of FNR, while the second half region contains three consecutive substitutions of ATCAA to ATTTT, and therefore does not contain a complete FNR binding site. The ArcA binding site contained therein is TTTAATTA, and compared with the conserved core binding site 5'-GTTAATTA-3' of ArcA, the first G is replaced by T. Therefore, the promoter of sodA contains only a single ArcA binding site, and its FNR binding site is incomplete, which does not meet the rules for the reverse hyperoxia promoter in the present invention.

[0126] In a preferred embodiment of the present invention, the hypoxia-specific gene expression cassette is regulated by oxygen concentration.

[0127] In order for gram-negative bacteria such as Salmonella to survive in the hypoxic zone, 1% oxygen concentration is a very important key point, which represents pathological hypoxia. Because oxygen concentrations below 1% are a clear sign of a tumor hypoxic zone, and there are no areas with oxygen concentrations below 1% in normal organ tissues. For example, the oxygen concentration in the hypoxic zone of pancreatic cancer, cervical cancer, prostate cancer and many other tumors is less than 0.7%.

[0128] The hypoxia-specific gene expression cassette designed by the present invention enables Salmonella and other Gram-negative bacteria to recognize hypoxic areas of tumors and proliferate in hypoxic areas of tumors by precisely regulating oxygen. The goal achieved by the present invention is that when the oxygen concentration is lower than 0.8%, the modified Salmonella and other Gram-negative bacteria can survive and proliferate, and achieve suicide lysis in a normal oxygen concentration environment.

[0129] Thus, in a more preferred embodiment of the present invention, the positive hypoxia promoter functions when the oxygen content is lower than 1%, but fails to function when the oxygen content is higher than 1%; and / or

[0130] The inverse hyperoxic promoter is functional when the oxygen content is above 1%, but ineffective when the oxygen content is below 1%.

[0131] 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

[0132] The inverse hyperoxic promoter is functional when the oxygen content is above 0.8%, but ineffective when the oxygen content is below 0.8%.

[0133] Another aspect of the present invention provides a method for controlling the expression of a drug protein in a prokaryotic cell using the expression vector of the present invention, which comprises the following steps:

[0134] 1. Prepare the expression vector of the present invention;

[0135] 2. Integrate the constitutively expressed bacteriophage RNA polymerase gene expression cassette into the host bacterial chromosome;

[0136] 3. Knock out the asd gene on the host bacterial chromosome;

[0137] 4. Transform the expression vector of the present invention into host bacteria.

[0138] 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.

[0139] In a preferred embodiment of the present invention, the host bacterium is a Gram-negative bacterium.

[0140] In the present invention, term " gram-negative bacteria " refers to after carrying out the known partial procedure of gram staining, the bacteria of the initial basic dye staining agent (such as, crystal violet) as is not retained.In exemplary gram staining, first by heating, cell is fixed on slide and dyed with basic dye (such as, crystal violet), and described basic dye is absorbed by both gram-negative bacteria and gram-positive bacteria.Then, slide is processed with mordant (such as, Gram's iodine solution), and described mordant is combined with basic dye (such as, crystal violet) and is trapped in cell.Then, cell is washed with acetone or ethanol, and then counterstained (such as, safranin) with the second dye of different colors.Gram-positive organism retains initial purple dyeing, and gram-negative organism is decolorized by organic washing solvent, therefore shows counterstaining. Exemplary Gram-negative bacteria include, but are not limited to, Escherichia species, Shigella species, Salmonella species, Campylobacter species, Neisseria species, Haemophilus species, Aeromonas species, Francisella species, Yersinia species, Klebsiella species, Bordetella species, Legionella species, Corynebacterium species, Citrobacter species, Chlamydia species, Brucella species, Pseudomonas species, Helicobacter species, and Vibrio species.

[0141] Thus, in a more preferred embodiment of the present invention, the Gram-negative bacteria are selected from the group consisting of Salmonella, Escherichia coli, Shigella, Yersinia, Enterobacter cloacae, Cronobacter, Klebsiella, Pantoea, Serratia, Simhuisella, Enterobacter reuteri, Haemophilus, Vibrio, Pseudomonas, Pasteurella, Burdetella, Bordetella pertussis, Acinetobacter baumannii, Burkholderia, Vibrio vulnificus, Bacteroides fragilis, Pseudomonas syringae, Pseudomonas putida , Legionella, Klebsiella pneumoniae, 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.

[0142] In the present invention, the term "live bacteria" refers to a strain that has vitality, active nutritional metabolic characteristics and can perform its own biological functions. Live bacteria can include bacterial biomass produced during the metabolism of the strain.

[0143] In a more preferred embodiment of the present invention, the Gram-negative bacteria is Salmonella.

[0144] When the expression vector of the present invention is transferred into Gram-negative bacteria (e.g., Salmonella typhimurium) as a therapeutic bacterium, the efficient expression of the drug protein gene and the membrane-breaking protein gene controlled by the specific phage RNA polymerase integrated into the bacterial chromosome and the promoter of the dual phage source will lead to increased toxicity of the bacteria. Therefore, in order to achieve the safety of therapeutic bacteria, the above-mentioned 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 toxicity island regulation defects.

[0145] Therefore, in a preferred embodiment of the present invention, the Gram-negative bacteria are attenuated Gram-negative bacteria.

[0146] In a more preferred embodiment of the present invention, the attenuated Gram-negative bacteria is Salmonella.

[0147] 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.

[0148] Another aspect of the present invention provides use of the expression vector of the present invention or the Gram-negative bacteria of the present invention in the preparation of tumor drugs.

[0149] In a preferred embodiment of the present invention, the tumor is a solid tumor.

[0150] The term "solid tumor" used in the present invention refers to an abnormal mass of tissue, which does not usually include cysts or liquid areas. Solid tumors may be benign (non-cancer), or malignant (cancer). Different types of malignant solid tumors are named after the cell types that form them. Examples of malignant solid tumors are sarcomas, carcinomas and lymphomas. Leukemia (blood cancer) does not usually form malignant solid tumors. Malignant solid tumors include but are not limited to abnormal cell masses that may be derived from different tissue types, such as liver, colon, colorectal, skin, breast, pancreas, cervix, uterine body, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testis, thyroid or lung, so malignant solid tumors include malignant solid liver tumors, colon tumors, colorectal tumors, skin tumors, breast tumors, pancreatic tumors, cervical tumors, uterine body tumors, bladder tumors, gallbladder tumors, kidney tumors, laryngeal tumors, lip tumors, oral tumors, esophageal tumors, ovarian tumors, prostate tumors, stomach tumors, testicular tumors, thyroid tumors or lung tumors, etc.

[0151] Thus, in a more preferred embodiment of the present invention, the solid tumor is selected from the group consisting of tumors / cancers of the breast, bone, liver, lung, skin, kidney, stomach, pancreas, prostate, lymph (non-Hodgkin's lymphoma, Hodgkin's lymphoma), intestine (colon cancer, rectal cancer), pelvic cavity (cervical cancer, ovarian malignancy, endometrial cancer, ovarian cancer), nervous system, head and neck cancer, and bladder.

[0152] 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.

[0153] In the present invention, for the purpose of treatment, the term "subject" is preferably a subject in need of treatment of a target pathological condition, such as a tumor. For the purpose of prevention, the subject is preferably a subject at risk of developing a target pathological condition or prone to developing a target pathological condition. The term "subject" includes living organisms, such as prokaryotes and eukaryotes. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, hedgehogs, rats, and transgenic non-human animals. In a specific embodiment of the present invention, the subject is a human.

[0154] As used herein, "treatment" is a process for obtaining a beneficial or desired clinical outcome. For purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing the proliferation of neoplastic or cancerous cells (or destroying neoplastic or cancerous cells), inhibiting the metastasis of neoplastic cells, shrinking or reducing the size of a tumor, alleviating a malignant tumor, alleviating symptoms caused by a malignant tumor, improving the quality of life of a subject with a malignant tumor, reducing the dose of other drugs required to treat a malignant tumor, delaying the progression of a malignant tumor, curing a malignant tumor, and / or prolonging the survival of a patient with a malignant tumor.

[0155] As used herein, an "effective amount" or "effective dose" of a bacterium, drug, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of disease, reducing the severity of disease, or delaying the onset of disease, including biochemical, histological, and / or behavioral symptoms of disease, its complications, and intermediate pathological phenotypes presented during the development of the disease. For therapeutic use, beneficial or desired results include, for example, alleviating one or more symptoms of a disease (such as a tumor), reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, prolonging the survival of the treated subject, and / or delaying the progression of cancer in a patient. For example, an "effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts the efficacy of the drug on human tumors. Alternatively, it can also be evaluated by examining the ability to inhibit cell growth, which can be determined in vitro by tests known to those skilled in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject. Such an amount can be determined by one skilled in the art based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0156] As used in the present invention, "pharmaceutically acceptable carrier" includes any material that allows the ingredient to retain biological activity when combined with the active ingredient and does not react with the subject's immune system, including but not limited to disintegrants, binders, fillers, buffers, tonicity agents, stabilizers, antioxidants, surfactants or lubricants. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion). For example, depending on the route of administration, the bacteria of the present invention can be wrapped in a material to protect the bacteria from the effects of acids and other natural conditions that can inactivate the bacteria. Pharmaceutically acceptable carriers include physiological saline, PBS buffer, sterile aqueous solutions or dispersions, and powders for the temporary preparation of injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any range that is incompatible with the active compound, may be in the pharmaceutical composition of the present invention.

[0157] 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.

[0158] In a preferred embodiment of the present invention, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

[0159] 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.

[0160] In a preferred embodiment of the present invention, the pharmaceutical composition is used to treat solid tumors. The bacteria, drugs or pharmaceutical compositions of the present invention are administered by the following routes: intravenous injection, intratumoral injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intracerebral administration, gastrointestinal administration, body surface administration, oral mucosal administration, nasal administration, rectal administration, or vaginal administration.

[0161] 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.

[0162] The dosage form of the drug or pharmaceutical composition of the present invention can be a solution, emulsion, freeze-dried preparation or suspension; for oral administration, the dosage form can be a tablet or capsule form; for intranasal dosage form, the dosage form can be a powder, nasal drops or aerosol form; for topical administration, the dosage form can be an aqueous solution, suspension, ointment, cream or gel; for rectal or vaginal administration, the dosage form can be a suppository, enema or delivered as part of an endoscopic or colonoscopy procedure.

[0163] Bacteria, medicine or pharmaceutical composition of the present invention can be manufactured by methods well known in the art, such as microbial growth in a fermentation tank, followed by centrifugal concentration and washing, filtration or dialysis, conventional granulation, mixing, dissolving, encapsulation, lyophilization or emulsification processes and other methods. Bacteria, medicine or pharmaceutical composition of the present invention can be produced in various forms, including particles, precipitation or microparticles, powders, including freeze-dried, rotary dried or spray-dried powders, amorphous powders, injections, emulsions, elixirs, suspensions or solutions. Preparations can optionally contain stabilizers, pH adjusting agents, surfactants, bioavailability regulators and these combinations.

[0164] Bacteria, medicine or pharmaceutical composition of the present invention can be used alone, or in the presence of a carrier, combined with other compounds or compositions. In a preferred embodiment of the present invention, the bacterium, medicine or pharmaceutical composition can be combined 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.

[0165] The present invention achieves the following beneficial effects by adopting the above technical solution:

[0166] 1. The present invention combines a phage-derived promoter with a specific phage RNA polymerase gene to make the two cooperate with each other, and uses a plasmid-mounted expression platform to achieve stable and efficient expression of drug proteins in the tumor area.

[0167] 2. The present invention combines a phage-derived promoter with a membrane-breaking protein gene and a drug protein gene to coordinate the three, thereby stably and efficiently delivering drugs and achieving efficient killing of tumor cells.

[0168] 3. The present invention rationally designs a hypoxia-specific gene expression cassette, so that the overall mutation rate of the modified bacteria is low, specific distribution is achieved inside the tumor, and the clearance rate in normal organs is faster. BRIEF DESCRIPTION OF THE DRAWINGS

[0169] Figure 1 Agarose gel electrophoresis identification of the positive hypoxia promoter yhbU-S and ynfK-S clone verification diagram.

[0170] Figure 2 Agarose gel electrophoresis was used to identify the essential gene library and verify the amplification pattern.

[0171] Figure 3 Agarose gel electrophoresis identification of reverse high oxygen promoter cyoA-S and ydcI-S cloning verification diagram.

[0172] Figure 4 Schematic representation of the hypoxia-specific gene expression cassette.

[0173] Figure 5 Schematic diagram of the lambda RED recombinase and CRE recombinase systems.

[0174] Figure 6 Figure 1. Knockout experiment identification of Salmonella aroA gene.

[0175] (A) Agarose gel electrophoresis to identify the PCR amplification product of the aroA gene knockout fragment;

[0176] (B) Verification of the PCR amplification product of the target fragment of strain SWT003 identified by agarose gel electrophoresis.

[0177] Figure 7 Figure 1. Knockout experiment identification of Salmonella alr gene.

[0178] (A) Agarose gel electrophoresis to identify the PCR amplification product of the alr gene knockout fragment;

[0179] (B) Verification of the PCR amplification product of the target fragment of strain SWT004 identified by agarose gel electrophoresis.

[0180] Figure 8 Agarose gel electrophoresis identification of strains SWT1001 and SWT1005 after the insertion of the hypoxia-specific gene expression cassette (alr is an essential gene).

[0181] Fig. 9 Verification of the PCR amplification product of SWT007 identified by agarose gel electrophoresis.

[0182] Fig.10 Agarose gel electrophoresis identification of strains SWT2009 and SWT2013 after the insertion of the hypoxia-specific gene expression cassette (dadX is an essential gene).

[0183] Fig.11 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1001.

[0184] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0185] (B) Aerobic culture on LB plates without D-alanine.

[0186] Fig.12 Figure 1. Results of oxygen adaptability validation test of Salmonella strain SWT1005.

[0187] (A) Cultured under anaerobic conditions on LB plates without D-alanine;

[0188] (B) Aerobic culture on LB plates without D-alanine.

[0189] Fig.13 Figure 1. Results of oxygen adaptability validation test of Salmonella strains SWT2009 and SWT2013.

[0190] (A) SWT2009 was cultured under anaerobic conditions on the upper plate and aerobic conditions on the lower plate on the LB plate without D-alanine.

[0191] (B) SWT2013 was cultured on an LB plate without D-alanine, with the upper plate showing anaerobic culture and the lower plate showing aerobic culture;

[0192] Fig.14 Figure 2 shows the oxygen adaptability test results of various Salmonella strains at oxygen concentrations below 0.8%.

[0193] (A) Salmonella strain SWT1001 was cultured on LB plates;

[0194] (B) Salmonella strain SWT1005 was cultured on LB plates;

[0195] Fig.15 Schematic diagram of pPRO005 containing the chloramphenicol resistance gene, lacUV5 and T7 RNA polymerase gene cassette.

[0196] Fig.16 Enzyme digestion identification diagram of plasmid pPRO005.

[0197] The arrow points to the excised T7 RNA polymerase gene cassette fragment.

[0198] Fig.17 Identification of the lacUV5-controlled T7 RNA polymerase constitutive expression cassette recombined into the Salmonella chromosome.

[0199] (A) PCR amplified fragment used for recombination (primers SWTO102 and SWTO103);

[0200] (B) PCR identification diagram of strains SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P after construction.

[0201] Fig.18 Schematic diagram of the construction of plasmid expression vector pPRO010.

[0202] Fig.19 PCR identification of plasmid pPRO010 in strains SWT5001, SWT5005, SWT6009, and SWT6013.

[0203] Fig. 20 Schematic diagram of the construction of plasmid expression vector pPRO012.

[0204] Fig.21 PCR identification of plasmid pPRO012 in strains SWT7001, SWT7005, SWT7009, and SWT7013.

[0205] Fig. 22 Western Blot was used to identify the expression of the target gene in the expression vector strain and the control vector strain under culture medium conditions.

[0206] (A) Expression of DTA in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013;

[0207] (B) Expression of LLO in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013;

[0208] (C) Expression of T7 RNA polymerase in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013.

[0209] Fig.23 Western Blot was used to identify the expression of target genes in tumors of mice containing expression vector strains and control vector strains.

[0210] (A) Expression of DTA in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013;

[0211] (B) Expression of LLO in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013;

[0212] (C) Expression of T7 RNA polymerase in strains SWT5001, SWT5005, SWT6009, SWT6013 and strains SWT7001, SWT7005, SWT7009, SWT7013.

[0213] Fig.24 Identification of mouse tumor sections after treatment with strain SWT5005.

[0214] The dark part indicated by the arrow is the antibody-positive staining area;

[0215] (A) Tumor sections stained with anti-Salmonella antibodies;

[0216] (B) Tumor sections stained with anti-HIF1α antibody;

[0217] (C) Tumor sections stained with anti-Diphtheria toxin antibody.

[0218] Fig.25 Microscope images of the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0219] (A) EMT6;

[0220] (B) K7M2;

[0221] (C)Hepa1-6.

[0222] Fig.26 Microscope images of the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0223] (D)A549;

[0224] (E) B16F10;

[0225] (F) Renca.

[0226] Fig. 27 Microscope images of the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0227] (G) MFC;

[0228] (H) Pan02;

[0229] (I)RM-1.

[0230] Fig.28 Microscope images of the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0231] (J) CT26;

[0232] (K) ID8;

[0233] (L) Neuro-2a.

[0234] Fig.29 Microscope images of the killing effects of strains SWT5001, SWT5005, SWT6009, and SWT6013 on cancer cells after co-culture with them under anaerobic conditions.

[0235] (M) SCC7;

[0236] (N)MB49.

[0237] Fig.30Figure 3. Killing effects of CCK8-expressing 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.

[0238] (A) SWT5001 and SWT7001;

[0239] (B) SWT5005 and SWT7005.

[0240] Fig.31 Figure 3. Killing effects of CCK8-expressing 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.

[0241] (A) SWT6009 and SWT7009;

[0242] (B) SWT6013 and SWT7013.

[0243] Fig.32 Identification of the inhibitory effects of the expression vector strain SWT5005 and the control vector strain SWT7005 in different mouse tumor models.

[0244] Vehicle: blank control, no drug treatment;

[0245] (A) EMT6;

[0246] (B) K7M2;

[0247] (C) Hepa1-6;

[0248] (D)A549.

[0249] Fig.33 Identification of the inhibitory effects of the expression vector strain SWT5005 and the control vector strain SWT7005 in different mouse tumor models.

[0250] Vehicle: blank control, no drug treatment;

[0251] (A) B16F10;

[0252] (B) Renca;

[0253] (C)MFC;

[0254] (D)Pan02.

[0255] Fig.34 Identification of the inhibitory effects of the expression vector strain SWT5005 and the control vector strain SWT7005 in different mouse tumor models.

[0256] Vehicle: blank control, no drug treatment;

[0257] (A) RM-1;

[0258] (B) CT26;

[0259] (C) ID8;

[0260] (D) Neuro-2a.

[0261] Fig.35 Identification of the inhibitory effects of the expression vector strain SWT5005 and the control vector strain SWT7005 in different mouse tumor models.

[0262] Vehicle: blank control, no drug treatment;

[0263] (A) SCC7;

[0264] (B)MB49. DETAILED DESCRIPTION

[0265] The present application is further described in detail below through the accompanying drawings and embodiments. Through these descriptions, the characteristics and advantages of the present application will become clearer and more specific.

[0266] Example 1: Construction of a hypoxia-specific gene expression cassette with alr and dadX genes as essential genes for survival

[0267] In this embodiment, the forward hypoxia promoter uses Salmonella yhbU (yhbU-S) (SEQ ID No. 31), the survival essential gene uses the alr gene of Salmonella (SEQ ID No. 27), the reverse hyperoxic promoters use Salmonella cyoA (cyoA-S) (SEQ ID No. 33) and Salmonella ydcI (ydcI-S) (SEQ ID No. 34), and the forward hypoxia promoter uses Escherichia coli ynfK (ynfK-E) (SEQ ID No. 32), the survival essential gene uses the dadX gene of Salmonella (SEQ ID No. 29), and the reverse hyperoxic promoters use 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.

[0268] The combination of hypoxia-specific gene expression cassettes is shown in Table 1.

[0269] Table 1 Combination table of forward hypoxia promoter and reverse hyperoxia promoter (wherein the essential survival gene is the alr gene or dadX gene of Salmonella)

[0270]

[0271] (I) Construction of positive hypoxia promoter clone library

[0272] 1. Pick a single clone of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Industrial Microbiological Culture Collection) or Escherichia coli DH10B (purchased from Shanghai Weidi Biotechnology Co., Ltd.) and inoculate it into 5 ml LB liquid culture medium, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours. OD 600 The reading is between 2-3;

[0273] 2. Use a pipette to draw 2 μl of the corresponding strain culture solution and mix it with the corresponding primers and high-fidelity PCR amplification enzyme (purchased from TAKARA) system, and then mix and put it into the PCR amplification device;

[0274] 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;

[0275] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0276] 4. Mix the recovered product with the endonuclease (purchased from NEB) digestion system and incubate at 37°C for 1 hour;

[0277] 5. Purify and recover the enzyme cleavage products through a DNA gel recovery system.

[0278] The primers and endonucleases used for different forward hypoxia promoters are as follows:

[0279] 1. yhbU-S (SEQ ID No. 31): strain SWT001 was amplified using primers SWTO19 and 20, and the product was double-cut with NotI and HindIII and recovered;

[0280] 2. ynfK-E (SEQ ID No. 32): amplified from E. coli strain DH10B using primers SWTO21, 22, and the product was double-cut with NotI and HindIII and recovered;

[0281] The results of the verification of the positive hypoxia promoter clone library by agarose gel electrophoresis are shown in the attached figure. Figure 1 shown.

[0282] (II) Construction of a library of genes essential for survival

[0283] 1. Pick a single clone of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Industrial Microbiological Culture Collection Management Center) and inoculate it into 5 ml LB liquid culture medium, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0284] 2. Use a pipette tip to absorb 2 μl of the corresponding strain culture medium and mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then mix and put it into the PCR amplification equipment;

[0285] 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;

[0286] 3. Purify and recover the amplified PCR product through a DNA gel recovery system;

[0287] 4. Mix the recovered product with the enzyme digestion system and incubate at 37 degrees for 1 hour;

[0288] 5. Purify and recover the enzyme cleavage products through a DNA gel recovery system.

[0289] The genes essential for survival from different sources are as follows:

[0290] 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;

[0291] 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;

[0292] The results of the verification of the survival essential gene library by agarose gel electrophoresis are shown in the attached Figure 2 shown.

[0293] (III) Construction of the reverse hyperoxia promoter library

[0294] 1. Pick a single clone of wild-type Salmonella typhimurium (strain SWT001, purchased from CICC China Industrial Microbiological Culture Collection Management Center) and inoculate it into 5 ml LB liquid culture medium, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0295] 2. Use a pipette to draw 2 μl of the corresponding strain culture solution and mix it with the corresponding primers and high-fidelity PCR amplification enzyme system, and then mix and place it in the PCR amplification device;

[0296] 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;

[0297] 3. The amplified PCR product is purified and recovered through a DNA gel recovery system;

[0298] 4. Mix the recovered product with the enzyme digestion system and incubate at 37°C for 1 hour;

[0299] 5. Purify and recover the enzyme cleavage products through a DNA gel recovery system.

[0300] The primers and endonucleases used for different reverse hyperoxia promoters are as follows:

[0301] 1. cyoA-S (SEQ ID No. 33): strain SWT001 was amplified using primers SWTO43 and 44, and the product was double-cut with XhoI and PstI and recovered;

[0302] 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;

[0303] The results of the verification of the reverse high oxygen promoter clone library by agarose gel electrophoresis are shown in the attached figure. Figure 3 shown.

[0304] (IV) Construction of a combinatorial library of positive hypoxia promoters, survival essential genes, and negative hyperoxia promoters

[0305] The composition of the hypoxia-specific gene expression cassette is shown in the attached Figure 4 As shown, it is composed of a positive hypoxia promoter, a survival essential gene and a negative hyperoxia promoter in sequence.

[0306] As shown in Table 1, a cloned library was formed by combining the sequences of the forward hypoxia promoter library digested by NotI and HindIII, the survival essential gene Salmonella alr gene (SEQ ID No.27) or Salmonella dadX gene (SEQ ID No.29) digested by HindIII and XhoI, and the reverse hyperoxia promoter library digested by XhoI and PstI, and ligated into the plasmid pSWT003 vector (vector backbone, pBlueScript SK (+), purchased from Biowind) digested by SpeI and PstI, and the plasmid pSWT007 vector (containing the double-sided same-direction loxp sequences and the DNA fragment of the chloramphenicol resistance gene (SEQ ID No.37)) digested by SpeI and NotI.

[0307] The products amplified by the primers for Salmonella SWT001 or Escherichia coli DH10B 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.

[0308] (V) Construction of the strain corresponding to the present invention

[0309] 1. Wild-type Salmonella typhimurium (SWT001)

[0310] Wild-type Salmonella typhimurium (SWT001) was purchased from China Industrial Microbiology Culture Collection Center (CICC).

[0311] 2. Construction of Salmonella (SWT002) containing temperature-induced lambda-RED recombinase and loxp-CRE enzyme system

[0312] 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.

[0313] Plasmid pSWT001 is attached Figure 5As shown, it contains a lambda-RED recombinase module (SEQ ID No.38) (similar in function to the plasmid vector psim6 of Biowind) and a loxp-Cre recombinase module (SEQ ID No.38) (similar in function to the plasmid vector 705-Cre of Gene Bridges). The lambda-RED recombinase module consists of three recombinases, EXO, BET, and GAM, and the recombinase is controlled by the CI857 temperature control regulator. Therefore, it cannot be expressed under the culture condition of 32°C, and it will only be expressed when it is higher than 37°C. Under temperature-induced conditions, the recombinase EXO cuts the 5' end of the double-stranded linear DNA to cause a single-stranded DNA protruding at the 3' end. The single-stranded DNA will be bound by the BET protein and protected from degradation by other nucleases. The role of GAM is to inhibit the action of endogenous nucleases in bacteria. The homologous arms of homologous recombination are about 35-50bp, which are added on both sides of the DNA fragment to be recombined by PCR. The advantage of this technology is that it can accurately target the target region of the bacterial chromosome without causing additional mutations. The outermost parts of the recombined double-stranded DNA fragments include homology arm sequences of the positions to be recombined, and inside are loxp sequences in the same direction on both sides, totaling 34bp. The sites of the double loxp in the same direction are chloramphenicol resistance genes, which are used to screen recombinant bacteria. After successful recombination, the CRE enzyme system carried on the pSWT001 plasmid (also controlled by the CI857 temperature control regulator) specifically recognizes the loxp sequence, and can cut the sequence in the middle of the loxp in the same direction, leaving a loxp sequence, thereby eliminating the chloramphenicol resistance gene. Repeating the above operation can achieve continuous knockout and knock-in of genes.

[0314] The specific steps are as follows:

[0315] (1) Streak strain SWT001 on an LB plate and culture it statically in a 37°C incubator overnight;

[0316] (2) Pick a single clone and inoculate it into 5 ml of LB liquid culture medium, and place it in a constant temperature shaker at 37°C and 220 rpm for 16 hours;

[0317] (3) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density grows to OD 600 =0.3 Place on ice and let stand for 1 hour;

[0318] (4) Wash the cells three times with sterilized pure water;

[0319] (5) The recovered cells were mixed with 10 ng of plasmid pSWT001 and electroporated at a voltage of 1.8 kV;

[0320] (6) The electroporated cells were spread on LB plates containing 100 μg / ml ampicillin sodium and cultured in a 32°C incubator overnight until a single clone colony was grown, which was named SWT002.

[0321] 3. Construction of attenuated Salmonella with aroA gene knockout (SWT003)

[0322] (1) A single clone of SWT002 was selected and inoculated into 5 ml of LB liquid culture medium containing 100 μg / ml ampicillin sodium, and placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0323] (2) Inoculate the culture into fresh LB liquid medium containing 100 μg / ml ampicillin sodium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0324] (3) Wash the cells three times with sterilized pure water.

[0325] (4) Preparation of PCR products of SWTO1 and SWTO2.

[0326] Primers SWTO1 and SWTO2 were mixed with plasmid pSWT002 (containing loxP sequences in the same direction on both sides and 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.

[0327] 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.

[0328] The amplified PCR products were verified by agarose gel electrophoresis for the PCR amplification products of SWTO1 and SWTO2 of the aroA gene knockout fragment. The verification results are shown in the attached Figure 6 As shown in A.

[0329] The PCR products were purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR products were measured by nanodrop.

[0330] (5) The recovered bacteria were mixed with 100 ng of PCR products of SWTO1 and SWTO2 and electroporated at a voltage of 1.8 kV.

[0331] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured in a 32°C incubator overnight until a single clone colony was grown.

[0332] (7) Positive clones were identified by colony PCR, and the insertion of chloramphenicol resistance gene was identified by using SWTO4, SWTO6, SWTO3 and SWTO5.

[0333] The target fragment PCR amplification product of strain SWT003 was verified by agarose gel electrophoresis. The verification results are shown in the attached Figure 6 As shown in B.

[0334] (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.

[0335] 4. Construction of alr gene knockout attenuated bacteria (SWT004)

[0336] (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.

[0337] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0338] (3) Wash the cells three times with sterilized pure water.

[0339] (4) Preparation of PCR products of SWTO70 and SWTO71.

[0340] Primers SWTO70 and SWTO71 were mixed with plasmid pSWT002 and a high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0341] 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.

[0342] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0343] (5) The recovered bacteria were mixed with 100 ng of PCR products of SWTO70 and SWTO71 and electroporated at a voltage of 1.8 kV.

[0344] The PCR amplification products of SWTO70 and SWTO71 of the alr gene knockout fragments were verified by agarose gel electrophoresis. The verification results are shown in the attached Figure 7 As shown in A.

[0345] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured in a 32°C incubator overnight until a single clone colony was grown.

[0346] (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.

[0347] The target fragment PCR amplification product of strain SWT004 was verified by agarose gel electrophoresis. The verification results are shown in the attached Figure 7 As shown in B (the primers used are SWTO72, SWTO5, SWTO6, and SWTO73).

[0348] (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.

[0349] Example 2: Integrating the hypoxia-specific gene expression cassette into the dadX gene locus on the chromosome of the SWT004 strain (destroying its function)

[0350] Construction of alr-deficient attenuated bacteria containing a hypoxia-specific gene expression cassette

[0351] 1. Pick a single clone of the alr gene-knockout attenuated bacteria SWT004 and inoculate it into 5 ml of fresh LB liquid culture medium, and place it in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0352] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0353] 3. Wash the bacteria 3 times with sterilized pure water.

[0354] 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, pOL1005, and a high-fidelity PCR amplification enzyme system and place them into a PCR amplification device.

[0355] The amplification program was 95°C for 2 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 120 seconds, 30 cycles; 72°C for 10 minutes; and 4°C for 5 minutes.

[0356] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0357] 5. Mix the recovered bacteria with 100 ng of PCR product and perform electroporation at a voltage of 1.8 kV.

[0358] 6. Spread the electroporated cells on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine, and culture them in a constant temperature incubator at 32°C overnight until a single clone colony grows. The resulting strains are SWT1001 and SWT1005.

[0359] 7. Inoculate the single clone into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and culture it in a constant temperature shaker at 37°C and 220 rpm for 16 hours to eliminate the chloramphenicol resistance gene through the action of CRE enzyme.

[0360] 8. Primers SWTO61, 20; SWTO75, 20 were used to identify clones by colony PCR. The identification results of strain SWT1001 (yhbU-S+alr+cyoA-S combination) and strain SWT1005 (yhbU-S+alr+ydcI-S combination) are shown in the attached figure. Figure 8 shown.

[0361] Example 3: Construction of a hypoxia-specific gene expression cassette with the dadX gene as a survival essential gene

[0362] Since there are two isozyme genes for alanine racemase in the genomes of bacteria such as Salmonella and Escherichia coli, namely alr gene and dadX gene, respectively, in order to verify the effect, the corresponding homologous genes dadX gene pOL2009 and pOL2013 were used for the necessary genes for survival of the above-mentioned hypoxia-specific gene expression cassette.

[0363] The hypoxia-specific gene expression cassette was integrated into the alr gene locus on the chromosome of the SWT007 strain.

[0364] 1. Construction of attenuated bacteria with dadX gene knockout (SWT007)

[0365] (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.

[0366] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0367] (3) Wash the cells three times with sterilized pure water.

[0368] (4) Preparation of PCR products of SWTO78 and SWTO79.

[0369] Primers SWTO78 and SWTO79 were mixed with plasmid pSWT002 (same as above, plasmid description), and a high-fidelity PCR amplification enzyme system and placed into a PCR amplification device.

[0370] 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.

[0371] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0372] (5) The recovered bacteria were mixed with 100 ng of PCR products of SWTO78 and SWTO79 and electroporated at a voltage of 1.8 kV.

[0373] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and cultured in a 32°C incubator overnight until a single clone colony was grown.

[0374] (7) Positive clones were identified by colony PCR. SWTO59, SWTO5, SWTO6, and SWTO87 were used to identify the insertion of the chloramphenicol resistance gene. Fig. 9 shown.

[0375] (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.

[0376] 2. Construction of defective attenuated bacteria containing a hypoxia-specific gene expression cassette and the essential gene for survival being the dadX gene of Salmonella

[0377] (1) A single clone of the dadX gene knockout attenuated bacteria SWT007 was selected and inoculated into 5 ml of LB liquid culture medium, and placed in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0378] (2) Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density grows to OD 600 =0.3, place the culture flask in a 42°C water bath, shake and culture for 15 minutes, and then place on ice for 1 hour.

[0379] (3) Wash the cells three times with sterilized pure water.

[0380] (4) Prepare PCR products for constructing a strain containing a hypoxia-specific expression cassette. Mix primers SWTO85 and SWTO86 with plasmids pOL2009, pOL2013 containing a hypoxia-specific gene expression cassette, and a high-fidelity PCR amplification enzyme system and place them into a PCR amplification device.

[0381] 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.

[0382] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0383] (5) The recovered bacteria were mixed with 100 ng of PCR product and electroporated at a voltage of 1.8 kV.

[0384] (6) The electroporated cells were spread on a plate containing 25 μg / ml chloramphenicol and 100 μg / ml D-alanine and cultured in a 32°C incubator overnight until a single clone colony was grown.

[0385] (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.

[0386] (8) The corresponding primers SWTO98, 22; SWTO99, 22 were used to identify clones by colony PCR. The identification results of strains SWT2013 (ynfK-E+dadX+ydcI-S) and strains SWT2009 (ynfK-E+dadX+cyoA-S) are shown in the attached figure. Fig.10 shown.

[0387] Example 4: Verification of oxygen adaptability of Salmonella strains SWT1001, SWT1005, SWT2009, SWT2013 containing hypoxia-specific gene expression cassettes

[0388] 1. Pick a single clone of strains SWT1001, SWT1005, SWT2009, and SWT2013 containing a hypoxia-specific expression cassette and inoculate it into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and place it in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0389] 2. After the culture is completed, dilute the strain 10 times and measure the absorbance (OD value) at 600nm.

[0390] 3. Calculate the volume of bacterial solution per 1OD of bacteria according to the following formula and add deionized water to 1ml.

[0391]

[0392] 4. Take 10 μl of the above bacterial solution and spot it on two LB plates without D-alanine for culture, mark it as "1" and repeat three times, mark it as "a", "b", and "c".

[0393] 5. After 10-fold gradient dilution, take 10 μl of the dilution solution and spot it on the above culture medium, marking it as "2".

[0394] 6. Repeat this 10-fold gradient dilution until the mark "8" is reached.

[0395] 7. One of the plates was cultured at 37°C in an anaerobic environment, and the other plate was cultured at 37°C in an atmospheric environment (21% oxygen concentration).

[0396] The results of the oxygen adaptability verification test of the strain containing the hypoxia specific expression cassette are shown in the attached Figure 11-13 The results show that the Salmonella strains SWT1001, SWT1005, SWT2009, and SWT1023 containing the hypoxia-specific gene expression cassette exhibit hypoxia regulation capabilities.

[0397] Example 5: Simulation of oxygen concentration in a strain library containing a hypoxia-specific gene expression cassette

[0398] By using anaerobic gas production bags to consume oxygen in the sealed culture tank, and using an oxygen meter to measure the oxygen concentration in the sealed culture tank, the oxygen concentration in the sealed culture tank can be fixed within a specific concentration range after using the anaerobic gas production bags for a certain period of time. The specific method is as follows:

[0399] The anaerobic gas production bag (brand: Japan Mitsubishi, item number: D-119) was combined and placed in a 7.0L sealed culture tank (brand: Japan Mitsubishi, item number: D-112), and an oxygen meter (brand: Meicheng Electrochemical, item number: OX-100A) was also placed in the tank.

[0400] The above anaerobic gas-generating bag combination was used to verify the growth of the genetically modified strains below 0.8% oxygen concentration.

[0401] 1. Verification of the adaptability of the strain library containing hypoxia-specific gene expression cassettes to oxygen concentrations below 0.8%

[0402] Since the pathological hypoxic zone of a tumor has an oxygen concentration below 1%, the present invention intends to prove that when the oxygen concentration is below 1% (or close to 1%), the Salmonella transformed with the hypoxia-specific gene expression cassette can still grow normally.

[0403] The oxygen concentration is regulated by the anaerobic gas production bag. After the anaerobic gas production bag is consumed for 1 hour, the oxygen meter reading shows that the oxygen concentration is 0.8-1%. Therefore, this experiment simulates the hypoxic environment inside the tumor, verifies the growth of Salmonella on the culture dish after the anaerobic gas production bag consumes the sealed culture tank for 1 hour, and simulates the growth of Salmonella strains containing hypoxia-specific gene expression boxes in the hypoxic environment inside the tumor.

[0404] The operation process of the spot plate experiment is as follows:

[0405] (1) A single clone of the strain containing the hypoxia-specific expression cassette was selected and inoculated into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and placed in a constant temperature shaker at 37°C and 200 rpm for 16 hours.

[0406] (2) After the culture is completed, the strain is diluted 10 times and the absorbance (OD value) at 600 nm is measured.

[0407] (3) Calculate the volume of bacterial solution per 1 OD of bacteria according to the following formula and add deionized water to 1 ml.

[0408]

[0409] (4) Take 10 μl of the above bacterial solution and spot it on an LB plate containing D-alanine for culture, label it as “1” and repeat three times, label them as “a”, “b” and “c”.

[0410] (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".

[0411] (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”.

[0412] (7) Repeat this 10-fold serial dilution until the mark “8” is reached.

[0413] (8) The two plates were incubated at 37°C in an environment of 0.8% oxygen.

[0414] As attached Fig.14 As shown, the present invention compares different strains (attached Fig.14 (A) SWT1001, Attachment Fig.14 (B) STW1005) was measured by measuring the growth of each strain on an LB plate without D-alanine added to determine the growth of each strain below 0.8% oxygen concentration. Since the culture medium without D-alanine added requires the corresponding Salmonella strain to rely on its own oxygen regulation system to grow normally, the results confirmed that strains containing a low oxygen specific expression cassette that can grow normally in an anaerobic environment can all grow normally below 0.8% oxygen concentration.

[0415] 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

[0416] (I) Construction of lacUV5-controlled T7 RNA polymerase constitutive expression plasmid pPRO005

[0417] To construct constitutively expressed T7 RNA polymerase, construct vector pPRO005. Fig.15 As shown, the constitutive expression promoter lacUV5 was placed upstream of the RBS sequence (SEQ ID No.35) T7 RNA polymerase (SEQ ID No.4), and the chloramphenicol resistance gene cm (SEQ ID No.37) with two loxp sequences on both sides was added upstream of lacUV5, in order to facilitate the next step of recombination into the Salmonella chromosome for screening.

[0418] 1. The plasmid pSWT007 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was digested with SpeI and NotI to recover a fragment of about 1100 bp containing the chloramphenicol resistance gene cm with loxp sequences on both sides;

[0419] 2. Direct annealing of primers SWTO100 and SWTO101 produced NotI and HindIII cuts, and recovered a fragment of approximately 70 bp, including the promoter lacUV5;

[0420] 3. Plasmid pPRO004 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was digested with HindIII and XhoI to recover a fragment of about 2690 bp including the T7 RNA polymerase gene;

[0421] 4. Cut plasmid pSWT003 (plasmid backbone) with SpeI and XhoI to recover a fragment of approximately 3000 bp;

[0422] 5. Pass the above 5 fragments through T 4 DNA ligase was used to connect the clones, transform DH10B competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and spread on ampicillin and chloramphenicol double-resistant LB plates, and single clones were selected and named pPRO005;

[0423] 6. Extract plasmid pPRO005, take 100 ng and mix with NotI and XhoI endonuclease system, incubate at 37℃ for 1 hour, and identify by agarose gel electrophoresis. Fig.16 shown.

[0424] (ii) The recombinant lacUV5-controlled T7 RNA polymerase constitutive expression cassette is integrated into the Salmonella chromosome

[0425] 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, and place them in a constant temperature shaker at 32°C and 220 rpm for 16 hours.

[0426] 2. Inoculate the culture into fresh LB liquid medium at a ratio of 1:100 and continue culturing for 2-3 hours until the bacterial density grows to OD 600 =0.3, put the culture bottle in a 42°C water bath, shake and culture for 15 minutes, and then place it on ice for 1 hour.

[0427] 3. Wash the bacteria 3 times with sterilized pure water.

[0428] 4. Prepare PCR products of SWTO102 and SWTO103.

[0429] Primers SWTO102 and SWTO103 were mixed with plasmid pPRO005 and a high-fidelity PCR amplification enzyme system and placed in a PCR amplification device.

[0430] 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.

[0431] The amplified PCR products were verified by agarose gel electrophoresis for the PCR amplification products of SWTO102 and SWTO103. The verification results are shown in the attached Fig.17 As shown in A.

[0432] The amplified PCR product was purified and recovered by a DNA gel recovery system, and the concentration and purity of the recovered PCR product were measured by nanodrop.

[0433] 5. Mix the recovered bacteria with 100 ng of PCR products of SWTO102 and SWTO103, and perform electroporation at a voltage of 1.8 kV.

[0434] 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 them in a constant temperature incubator at 32°C overnight until a single clone colony grows.

[0435] 7. Inoculate the single clone into 5 ml 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.

[0436] 8. Identify positive clones by colony PCR, using SWTO104, SWTO106, SWTO105 and SWTO107 for identification.

[0437] 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 the attached Fig.17 As shown in B.

[0438] Example 7: Construction of strains carrying the three-in-one expression vector pPRO010 (SWT5001, SWT5005, SWT6009, SWT6013)

[0439] In this example, a plasmid vector without antibiotic resistance selection was constructed, as shown in the attached Fig.18As shown, pUC replicon (SEQ ID No.36) and Salmonella asd gene (SEQ ID No.11) were used as balanced lethal control mechanisms, and T7 promoter containing double phage sources was used to control the expression of drug protein exotoxin DTA fragment (SEQ IDNo.15) and membrane-breaking protein gene LLO (SEQ ID No.13). The constructed plasmid pPRO010 was transferred into SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, SWT2013-T7P, and the corresponding strains SWT5001, SWT5005, SWT6009, and SWT6013 were generated.

[0440] The specific steps are as follows:

[0441] 1. Cut plasmid pPRO006 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) with XhoI and NotI to recover a fragment of about 820 bp containing the pUC replicon;

[0442] 2. The plasmid pPRO007 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was digested with PstI and XhoI to recover a fragment of about 1270 bp containing the Salmonella asd gene;

[0443] 3. The plasmid pPRO008 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was digested with PstI to recover a fragment of about 660 bp containing the codon-optimized gene DTA of Salmonella controlled by the T7 promoter;

[0444] 4. The plasmid pPRO009 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was digested with NotI and PstI to recover a fragment of about 1660 bp containing the Salmonella codon-optimized gene LLO controlled by the T7 promoter;

[0445] 5. Pass the above 4 fragments through T 4 DNA ligase was used to transform competent cells of SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P, and 100 μg / ml D-alanine LB plates were coated, and single clones were selected and named SWT5001, SWT5005, SWT6009, and SWT6013.

[0446] 6. Pick out the single clones respectively, and amplify them with 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 identify them by agarose gel electrophoresis, as shown in the attached Fig.19 shown.

[0447] Example 8: Construction of control strains (SWT7001, SWT7005, SWT7009, SWT7013) carrying the three-in-one expression vector pPRO012

[0448] As a control strain, the present invention constructs an expression vector replacing the T7 promoter, using the promoter of the sseA gene on the pathogenicity island II of Salmonella (SEQ ID No. 40).

[0449] The promoter of sseA 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 the attached Fig. 20 As shown, pUC replicon (SEQ ID No.36) and Salmonella asd gene (SEQ ID No.11) were used as balanced lethal control mechanisms, and sseA promoter was used to control the expression of drug protein exotoxin DTA fragment (SEQ ID No.15) and membrane-breaking protein gene LLO (SEQ ID No.13). The constructed plasmid pPRO012 was transferred into SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, SWT2013-T7P, and the corresponding strains SWT7001, SWT7005, SWT7009, and SWT7013 were generated.

[0450] The specific steps are as follows:

[0451] 1. Cut plasmid pPRO010 with PstI and NotI to recover a 2050 bp fragment containing the pUC replicon and asd gene;

[0452] 2. The plasmid pPRO008 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was digested with NdeI and PstI to recover a 590 bp fragment containing the Salmonella codon-optimized gene DTA;

[0453] 3. The plasmid pPRO009 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was digested with NdeI and PstI to recover a fragment of about 1600 bp containing the Salmonella codon-optimized gene LLO;

[0454] 4. Plasmid pPRO011 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was digested with NotI and NdeI to recover a 425 bp fragment containing the sseA promoter and RBS site sequence, which was used as the promoter of LLO;

[0455] 5. Plasmid pPRO011 (synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.) was amplified using primers SWTO126 and SWTO127, and a 425 bp fragment containing the sseA promoter and RBS site sequence was recovered, and then recovered using PstI and NdeI. This fragment was used as the promoter of DTA.

[0456] 6. Pass the above 5 fragments through T 4 DNA ligase was used to transform competent cells of SWT1001-T7P, SWT1005-T7P, SWT2009-T7P, and SWT2013-T7P, and 100 μg / ml D-alanine LB plates were coated, and single clones were selected and named SWT7001, SWT7005, SWT7009, and SWT7013.

[0457] 7. Pick out the single clones respectively, amplify them with primers SWTO111 and SWTO128, and identify them by agarose gel electrophoresis. Fig.21 shown.

[0458] Example 9: Verification of the expression of T7 RNA polymerase, DTA, and LLO proteins in strains carrying expression vectors and control vectors

[0459] This example was designed to demonstrate that the protein regulated by the T7 promoter has stable expression in a culture environment and a tumor environment.

[0460] (I) Verification of protein expression in culture medium of expression vector strains SWT5001, SWT5005, SWT6009, SWT6013 and control vector strains SWT7001, SWT7005, SWT7009, SWT7013

[0461] 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 constant temperature incubator overnight.

[0462] 2. Pick out the strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 single clones 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.

[0463] 3. Take 1 ml of bacterial suspension of each strain, 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 at 95°C for 10 minutes, centrifuge again at 12000 RPM, and recover the supernatant for later use.

[0464] 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, catalog number C631100-0200), and perform electrophoresis at 120 V for 60 minutes.

[0465] 5. Transfer the SDS-PAGE gel after electrophoresis to a PVDF membrane (purchased from Roche, catalog number 03010040001) at a steady current of 250 mA for 30 minutes.

[0466] 6. Transfer the transferred PVDF membrane to 5% skimmed milk powder, place it on a horizontal shaker at 80 rpm, and incubate at room temperature for 1 hour as a blocking method.

[0467] 7. The blocked PVDF membrane was diluted with 5% skim milk powder and incubated at 4°C for 18 hours using 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.

[0468] 8. Use 5% skimmed milk powder to dilute the secondary antibody, transfer the PVDF membrane after the primary antibody incubation to the secondary antibody dilution solution, place it on a horizontal shaker, 80 rpm, and incubate at room temperature for 1 hour.

[0469] 9. Wash the PVDF membrane after incubation with secondary antibody with TBST Buffer (purchased from Sangon Biotechnology, catalog number C520009-0005) for 3 times, 5 minutes each time. Add ECL developer (developer SignalFire TM ECLReagent was purchased from CST, catalog number 6883p3), and incubated for 1 min in the dark. After incubation, the PVDF membrane was placed in a chemiluminescence instrument for image acquisition.

[0470] 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 Appendix Fig. 22 A; For LLO expression, see Appendix Fig. 22 B; T7 RNA polymerase expression see Appendix Fig. 22 C.

[0471] (II) Verification of protein expression of expression vector strains SWT5001, SWT5005, SWT6009, SWT6013 and control vector strains SWT7001, SWT7005, SWT7009, SWT7013 in the mouse CT26 tumor model

[0472] This example was designed to demonstrate that the T7 expression vector can stably express the target protein in the in vivo tumor environment.

[0473] Tumor model mice were injected with the corresponding expression vectors and control vector strains through the tail vein, and 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.

[0474] The specific steps are as follows:

[0475] 1. Establishment of tumor model

[0476] BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, raised in an SPF environment) were subcutaneously inoculated with 1×10 6 CT26 mouse colon cancer cells were used to establish a mouse colon cancer subcutaneous tumor model. 10-12 days after inoculation, the tumor volume was 100mm 3 The mice were divided into 8 groups, namely SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013, with 2 mice in each group. The tumor-bearing mice were inoculated with 1×10 7 CFU bacteria.

[0477] 2. Bacterial Preparation

[0478] Taking strain SWT1005 as an example, it was streaked on an LB plate to which D-alanine was additionally added, and was statically cultured in a 37°C constant temperature incubator overnight; strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 monoclonal clones were picked and inoculated into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and placed in a constant temperature shaker at 37°C and 220 rpm for culture for 16 hours; the absorbance (OD value) of the strain at 600 nm was measured, the culture with an OD value of 1 was calculated and measured, washed three times with PBS, prepared into a PBS bacterial suspension, and administered through the tail vein of mice.

[0479] 3. Tumor Extraction Sample Preparation

[0480] (1) On the third day after administration, mice were killed and tumor tissues were isolated. The tumor tissues were 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, and then resuspended in lysis buffer (protein loading buffer, purchased from CST, catalog number 7722S), heated at 95°C for 10 minutes, and centrifuged again at 12,000 rpm to recover the supernatant for later use.

[0481] (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, catalog number C631100-0200), and electrophoresed at 120 V for 60 minutes.

[0482] (3) The SDS-PAGE gel after electrophoresis was transferred to a PVDF membrane (purchased from Roche, catalog number 03010040001) at a constant current of 250 mA for 30 minutes.

[0483] (4) The PVDF membrane after transfer was transferred to 5% skimmed milk powder and placed on a horizontal shaker at 80 rpm and incubated at room temperature for 1 hour as a blocking method.

[0484] (5) 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).

[0485] (6) Dilute the secondary antibody with 5% skimmed milk powder, transfer the PVDF membrane after the 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.

[0486] (7) Wash the PVDF membrane after incubation with the secondary antibody three times with TBST Buffer (purchased from Sangon Biotechnology, catalog number C520009-0005), 5 minutes each time. Add ECL developer (developer SignalFire TM ECLReagent was purchased from CST, catalog number 6883p3), and incubated in the dark for 1 min. After incubation, the PVDF membrane was placed in a chemiluminescence instrument for image acquisition.

[0487] In the isolated tumor tissues, strains SWT5001, SWT5005, SWT6009, and SWT6013 identified by Western Blot can all express DTA at high intensity, see Appendix Fig.23 A; For LLO expression, see Appendix Fig.23 B; T7 RNA polymerase expression see Appendix Fig.23 C. However, only the expression of T7 RNA polymerase could be detected in the control strains SWT7001, SWT7005, SWT7009, and SWT7013. Fig.23 C, while almost no stable expression of DTA and LLO was detected.

[0488] Example 10: Immunohistochemical analysis of tumor area after injection of strain carrying expression vector

[0489] In order to verify that the Salmonella carrying the expression vector aggregates in the hypoxic area of ​​the tumor and expresses the target protein, this example selects SWT5005 as an example, and the mouse tumor model injected with Salmonella through the tail vein is sliced ​​and stained to observe the distribution of the drug protein in the tumor model.

[0490] The specific steps are as follows:

[0491] 1. Establishment of tumor model

[0492] BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, raised in an SPF environment) were subcutaneously inoculated with 1×10 6 CT26 mouse colon cancer cells were used to establish a mouse colon cancer subcutaneous tumor model. 10-12 days after inoculation, the tumor volume was 100mm 3 The mice were divided into PBS group and SWT5005 group, with 5 mice in each group. The tumor-bearing mice were inoculated with 1×10 7 CFU bacteria.

[0493] 2. Bacterial Preparation

[0494] Taking strain SWT5005 as an example, streak on LB plate with additional 100 μg / ml D-alanine, and culture statically in a 37°C constant temperature incubator overnight; pick a single clone of strain SWT5005 and inoculate it into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and place it in a constant temperature shaker at 37°C, 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 3 times with PBS, prepare a bacterial suspension in PBS, and administer it through the tail vein of mice.

[0495] 3. Analysis of tumor sample sections

[0496] On the 5th day after administration, the mice were killed, the tumor tissues were separated and immersed in formalin fixative; gradient dehydration was performed, the tissues were embedded in paraffin, and 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 3 times; the sections after antigen retrieval were immersed in 3% hydrogen peroxide solution for 10 minutes, and then rinsed with PBS 3 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 3 times; anti-Salmonella (Salmonella identification marker, purchased from abcam, catalog number ab35156), anti-HIF1α (hypoxic zone identification marker, purchased from abcam, catalog number ab51608), anti-diphtheria The primary antibody incubation solution of toxin (DTA identification antibody, purchased from abcam, catalog number ab151222) was added to different blocked tissue sections, incubated at 4°C overnight, and then rinsed with PBS three times; the secondary antibody incubation solution was added to the tissue sections, incubated for 25 minutes, and then rinsed with PBS three times; the DAB staining solution was added to the tissue sections, and counterstained with hematoxylin for 3 minutes; the tissue sections were dehydrated in a gradient manner, and after dehydration, the sections were sealed with sealing agents, and the sections were observed under a microscope and photographed, as shown in the attached Fig.24 shown.

[0497] The results showed that after antibody staining comparison, the brown (dark) area was antibody-positive, and the aggregation of Salmonella containing the hypoxia-specific gene expression cassette in the hypoxic area of ​​the tumor was found. Fig.24 As shown by the arrows in A and 24B, the expressed drug DTA was found to be widely distributed in the tumor, as shown in the attached Fig.24 Indicated by the arrow in C.

[0498] Example 11: Verification of the killing effect of strains carrying expression vectors and control vectors on cancer cells under hypoxic conditions

[0499] By co-culturing with cancer cells under hypoxic conditions in vitro, the killing effects of strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 on various cancer cells were verified.

[0500] The specific steps are as follows:

[0501] 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 constant temperature incubator overnight.

[0502] 2. Pick strains SWT5001, SWT5005, SWT6009, SWT6013 and SWT7001, SWT7005, SWT7009, SWT7013 single clones and inoculate them into 5 ml of fresh LB liquid culture medium containing 100 μg / ml D-alanine, and place them in a constant temperature shaker at 37°C and 220 rpm for 16 hours.

[0503] 3. Measure the absorbance of the strain at 600nm, take the culture with an OD value of 1, wash it twice with PBS, wash it once with the corresponding cell culture medium, and then resuspend it in the cell culture medium (the infection ratio is 200:1).

[0504] 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, and the cells were washed 3 times with PBS containing gentamicin. After changing the medium, the cells were cultured in an anaerobic tank at an oxygen concentration below 0.8% for 24 hours, and photographed with a 20x objective lens under the field of view of white light, as shown in the attached figure. Figure 25-29 shown.

[0505] 5. Mix CCK8 reagent with the above cell suspension and incubate for 1 hour.

[0506] 6. Use an enzyme-labeled instrument to measure the absorbance (detection wavelength 450 nm) and calculate the percentage of cytotoxicity.

[0507] 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 the attached figure. Fig.30 , as shown in 31.

[0508] The experimental results were analyzed using T test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0509] 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 through 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 the Salmonella carrying the three-in-one drug expression vector of the T7 expression system has universal killing effects on cancer cells.

[0510] Example 12: Evaluation of tumor suppressive properties of strains carrying expression vectors and control vectors in mouse tumor models

[0511] In order to verify the inhibitory effect of strains carrying expression vectors on mouse tumor growth, the present invention designed SWT5005 and SWT7005 as representatives for inhibitory experiments in mouse EMT6, K7M2, Hepa1-6, A549, B16F10, Renca, MFC, Pan02, RM-1, CT26, ID8, Neuro-2a, SCC7 and MB49 tumor models.

[0512] 1. Establishment of various mouse tumor models

[0513] (1) Establishment of EMT6 tumor mouse model

[0514] BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 EMT6 cancer cells were used to establish a mouse breast subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 100mm 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 with 1×10 7 CFU bacteria.

[0515] (2) Establishment of K7M2 tumor mouse model

[0516] BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 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 with 1×10 7 CFU bacteria.

[0517] (3) Establishment of Hepa1-6 tumor mouse model

[0518] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen free (SPF) environment) were subcutaneously inoculated with 1×10 6 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 with 1×10 7 CFU bacteria.

[0519] (4) Establishment of A549 tumor mouse model

[0520] Nu / Nu nude mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 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 with 1×10 7 CFU bacteria.

[0521] (5) Establishment of B16F10 tumor mouse model

[0522] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen free (SPF) environment) were subcutaneously inoculated with 1×10 6 B16F10 cancer cells were used to establish a mouse breast subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 100mm 3The mice were divided into PBS control group, SWT5005 and SWT7005 groups, with 5 mice in each group. The tumor-bearing mice were inoculated with 1×10 7 CFU bacteria.

[0523] (6) Establishment of Renca tumor mouse model

[0524] BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Renca cancer cells were used to establish a subcutaneous tumor model in mice. 14-18 days after inoculation, the tumor volume was 100mm 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 with 1×10 7 CFU bacteria.

[0525] (7) Establishment of MFC tumor mouse model

[0526] BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 MFC cancer cells were used to establish a mouse 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 with 1×10 7 CFU bacteria.

[0527] (8) Establishment of Pan02 tumor mouse model

[0528] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen free (SPF) environment) were subcutaneously inoculated with 1×10 6 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 with 1×107 CFU bacteria.

[0529] (9) Establishment of RM-1 tumor mouse model

[0530] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen free (SPF) environment) were subcutaneously inoculated with 1×10 6 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 with 1×10 7 CFU bacteria.

[0531] (10) Establishment of CT26 tumor mouse model

[0532] BALB / c mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 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 with 1×10 7 CFU bacteria.

[0533] (11) Establishment of ID8 tumor mouse model

[0534] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen free (SPF) environment) were subcutaneously inoculated with 1×10 6 ID8 cancer cells were used to establish a mouse 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 with 1×10 7 CFU bacteria.

[0535] (12) Establishment of Neuro-2a tumor mouse model

[0536] A / J mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 Neuro-2a cancer cells were used to establish a mouse subcutaneous tumor model. 14-18 days after inoculation, the tumor volume was 100mm 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 with 1×10 7 CFU bacteria.

[0537] (13) Establishment of SCC7 tumor mouse model

[0538] Nu / Nu nude mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen-free (SPF) environment) were subcutaneously inoculated with 1×10 6 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 with 1×10 7 CFU bacteria.

[0539] (14) Establishment of MB49 tumor mouse model

[0540] C57BL / 6 mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., weighing about 18 g, and raised in a specific pathogen free (SPF) environment) were subcutaneously inoculated with 1×10 6 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 with 1×10 7 CFU bacteria.

[0541] 2. Bacterial preparation and treatment

[0542] The constructed Salmonella strains SWT5005 and SWT7005 were selected and inoculated into 5 ml of LB liquid medium supplemented with D-alanine, and placed in a constant temperature shaker at 37°C and 220 rpm for 16 hours; 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 counting bacteria were administered to model mice; the day of administration was designated as day 0, and the length and width of the mouse tumor were measured on days 1, 3, 5, 7, 9, and 11 of administration, and the mouse tumor volume was calculated according to the following formula.

[0543] Mouse tumor volume = length × width 2 ×0.52

[0544] Compared with SWT5005 and SWT7005, the tumor inhibitory effects in mouse tumor models are shown in the attached figure. Figure 32-35 shown.

[0545] The experimental results were analyzed using T test, and p<0.05 (*), p<0.01 (**), and p<=0.001 (***) indicated significant differences.

[0546] 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.

[0547] The information of the corresponding strains of the present invention is shown in Table 2.

[0548] Table 2 Strain construction table of the present invention

[0549]

[0550]

[0551] The information of the corresponding tool plasmids of the present invention is shown in Table 3.

[0552] Table 3 Tool plasmid information table used in the present invention

[0553]

[0554]

[0555] The sequences of promoter, gene coding region, protein, etc. used in the present invention are shown in Table 4.

[0556] Table 4 Specific sequences of promoters, gene coding regions, proteins, etc. used in the present invention

[0557]

[0558]

[0559]

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567]

[0568]

[0569]

[0570]

[0571]

[0572]

[0573]

[0574]

[0575]

[0576]

[0577]

[0578]

[0579] The specific sequences of all primers used in the present invention are shown in Table 5.

[0580] Table 5 Specific sequences of all primers used in the present invention

[0581]

[0582]

[0583]

[0584]

[0585]

[0586] The present application has been described above in conjunction with preferred embodiments, but these embodiments are only exemplary and serve only as an illustration. On this basis, various replacements and improvements may be made to the present application, all of which fall within the scope of protection of the present application.

Claims

1. A 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) knocking 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.

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