SifA gene knockout attenuated salmonella recombinant engineering bacterium as well as preparation method and application thereof

Abstract

The invention discloses a sifA gene knockout attenuated salmonella recombinant engineering bacterium as well as a preparation method and application thereof, and belongs to the technical field of microbial biology. An intracellular vesicle (SCV) of the attenuated salmonella VNP20009 of the engineering bacterium forms a related gene sifA, and the related gene sifA is seamlessly knocked out. The engineering bacterium is named as VNP20009 [delta] sifA. The invention also discloses a preparation method of the engineering bacterium. The engineering bacterium has the characteristic of keeping the characteristic of specific colonization of a female parent strain VNP20009 in a tumor hypoxic region, and meanwhile, the biotoxicity of the strain is further reduced; meanwhile, the strain has a potential value of becoming a chassis strain for combined synthesis of biological tumor therapy due to relatively weak killing performance and higher intracellular load on immune cells. The invention also provides a construction method of the strain and application of the strain in treatment of mouse entity subcutaneous melanoma.

Description

Technical Field

[0001] The present invention relates to the field of microbial biotechnology, and particularly to an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout, a preparation method thereof, and an application thereof. Technical Background

[0002] The pathogenesis of Salmonella typhimurium highly depends on two different type III secretion systems (T3SS), which are encoded by Salmonella pathogenicity island 1 (SPI-1) and pathogenicity island 2 (SPI-2) respectively, and are crucial for the invasion and intracellular survival of Salmonella typhimurium (Nat Rev Microbiol, 2015, 13(4), 191-205.). SPI1 exists in all subspecies and serotypes of Salmonella and is essential for invading non-phagocytic cells. The activity of SPI2-T3SS is crucial for the intracellular lifestyle and systemic pathogenesis of Salmonella, and most SPI2-T3SS-deficient strains are detected with virulence defects in animal infection models (Cellular Microbiology, 2015, 17(5), 639-647. Mol Microbiol, 1998, 30(1), 163-174.). The survival of Salmonella in macrophages is a prerequisite for establishing systemic infection, and it mainly establishes systemic infection by replicating in host macrophages through the T3SS and virulence factors encoded by SPI-2 (Nat Commun, 2021, 12(1), 879.). When Salmonella enters macrophages through SPI1-dependent invasion or host cell-mediated phagocytosis, it avoids being killed by immune cells by forming a Salmonella-containing vacuole (SCV) and survives and replicates in the SCV. In the middle and late stages of SCV formation, intracellular acidification provides an important signal for the expression and secretion of SPI-2 effector molecules (Elife, 2023, 12, e85690.). The T3SS encoded by SPI2 secretes effector proteins that cross the SCV membrane and are released into the host cell cytosol, which is crucial for SCV vacuole modification and microtubule movement (Current opinion in microbiology, 2008, 11(1).). At the same time, these effector molecules promote the growth and extension of Salmonella-induced filaments (SIFs) from the SCV, providing space and nutrients for the growth of bacteria in the SCV (Cell Host & Microbe, 2017, 21(3), 390-402.). Among them, the effector protein SifA plays a key role in maintaining the integrity of the Salmonella-containing vacuole (SCV). Salmonella lacking the sifa gene cannot form a complete SCV and is free in the cytosol of RAW264.7 cells and cannot replicate (EMBO J, 2000, 19(13), 3235-3249.). On the other hand, SifA is a necessary condition for the formation of SIFs (Cellular Microbiology, 2001, 3(2), 75-84.).The linker region of the caspase-3 cleavage motif separates the two functional domains of SifA. The N-terminal domain of SifA binds to the SifA-kinesin interacting protein (SKIP), and the C-terminal domain contains a WxxxE motif that may mimic or activate the RhoA family GTPase (Cell Host Microbe, 2008, 4 (5), 434-446. Gut Microbes, 2019, 10 (2), 172-187.). These functional domains cooperate with another SPI-2 effector SseJ to promote the formation of SIFs (Mol Microbiol, 2002, 44 (3), 645-661. PLoS Pathog, 2023, 19 (9), e1011686.). The loss of the sifA gene will affect the ability of Salmonella to maintain the integrity of the SCV and the formation of SIFs.

[0003] As one of the most widely studied bacteria in bacterial-mediated cancer therapy, Salmonella typhimurium has tumor targeting, intrinsic oncolytic ability and immunomodulatory effects, making it an ideal carrier for drug and gene delivery (Acta Biochim Biophys Sin (Shanghai), 2023, 55 (3), 331-342.). However, as the most common pathogen, wild Salmonella typhimurium is obviously not feasible for direct cancer treatment. Constructing attenuated, controllable and effective engineered bacteria is a common transformation strategy. VNP20009 is a Salmonella typhimurium strain with msbB and purI genes deleted. Compared with the wild type, it significantly reduces the degree of host immune activation and improves tumor targeting. Its safety and efficacy have been demonstrated in animal tumor models. However, clinical trials have shown that its efficacy is not ideal, and additional research is needed to reduce dose-related toxicity and improve tumor localization (JCO, 2002, 20 (1), 142-152.). The effect of relying solely on bacteria to kill tumors is limited. Modifying bacteria to produce therapeutic molecules in tumors can increase the natural anti-tumor effect. For example, VNP20009 expressing FADD (Fas-associated protein with death domain) or N-FADD (FADD with truncated C-terminal tail) has a higher inhibitory effect on melanoma growth than VNP20009 (Sci Rep. 2016, 6, 34178.). However, in most cases, therapeutic molecules released from intracellular bacteria cannot pass through the bacterial envelope and SCV and be delivered to the cytosol, which greatly limits the effect of the drug (Sci Rep. 2016, 6, 30591.). The intracellular survival mode of bacteria after the loss of the sifA gene of Salmonella mentioned above just eliminates this limitation. On the other hand, Salmonella with sifA loss cannot replicate in macrophages, avoiding excessive proliferation in the cell, ensuring the survival of immune cells, and weakening the excessive immune response caused by the excessive toxicity of VNP20009.

[0004] Currently, there is a lack of an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout, as well as its preparation method and application. Summary of the Invention

[0005] The purpose of this application is to provide an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout, as well as its preparation method and application, which have good safety.

[0006] To solve the existing technical problems, the present invention provides the following technical solutions: In the first aspect, this application provides an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout.

[0007] In the second aspect, this application provides a bacterial agent of an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout.

[0008] In the third aspect, this application provides a preparation method of an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout.

[0009] In the fourth aspect, this application provides an application of an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout in the preparation of anti-tumor drugs.

[0010] In the fifth aspect, this application provides an application of an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout combined with other anti-tumor drugs in the preparation of anti-tumor drugs.

[0011] In the first aspect of this application, an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout is provided. The Salmonella recombinant engineering bacterium is an attenuated Salmonella recombinant engineering bacterium with sifA gene knockout derived or genetically modified based on attenuated Salmonella typhimurium VNP20009.

[0012] Furthermore, the attenuated Salmonella recombinant engineering bacterium with sifA gene knockout is a derivative engineering strain of engineered Salmonella VNP20009 with sifA gene deletion. The sifA gene of the parental strain VNP20009 is deleted. This derivative strain can specifically colonize in the core region of solid tumors; it has low toxicity to immune cells and high macrophage intracellular loading capacity; its LD50 for mice is more than 9 times higher than that of its parental strain VNP20009.

[0013] The derivative engineering bacterium of engineered Salmonella VNP20009 with sifA gene deletion can specifically colonize in the core region of solid tumors including melanoma, while maintaining low toxicity to immune cells; the complete nucleotide sequence of the genomic region from potB to the downstream gene potD is as shown in SEQ NO.2.

[0014] The second aspect of the present application provides a bacterial agent of attenuated Salmonella recombinant engineered bacteria with sifA gene knockout.

[0015] Furthermore, its active ingredient is at least one of the following (a), (b), (c), and (d):

[0016] (a) Attenuated Salmonella recombinant engineered bacteria cells;

[0017] (b) Ultrasonic disruption precipitate of attenuated Salmonella recombinant engineered bacteria;

[0018] (c) Primary extract of the fermentation broth of the obtained attenuated Salmonella recombinant engineered bacteria culture;

[0019] (d) Ultrasonic lysis supernatant of the obtained attenuated Salmonella recombinant engineered bacteria cells.

[0020] Furthermore, the preparation of the attenuated Salmonella recombinant engineered bacteria with sifA gene knockout includes oral preparations, injection preparations, intravenous preparations, or spray preparations.

[0021] The third aspect of the present application provides a method for preparing attenuated Salmonella recombinant engineered bacteria with sifA gene knockout, which is characterized by including the following steps:

[0022] (1) Construction of the sifA knockout vector pTAT-sifA-KO plasmid: Design sgRNA, then amplify the upstream and downstream fragments of sifA by PCR, and use one-step seamless cloning into the pTAT vector; After homologous recombination, chemically transform it into Escherichia coli TOP10; After sequencing, the nucleotide sequence of this vector is shown in SEQ NO.1;

[0023] (2) Electroporate pTAT-sifA-KO into Salmonella VNP20009 containing the pCas9 plasmid, culture for gene knockout, and then verify by agarose gel electrophoresis;

[0024] (3) Eliminate the plasmid of the correctly edited strain, use IPTG induction to eliminate the pTAT-sifA-KO plasmid, and then culture at 42°C to eliminate the pCas9 plasmid to obtain the attenuated Salmonella recombinant engineered bacteria with sifA gene knockout.

[0025] Furthermore, the genotype and phenotype of sifA gene deletion can be stably inherited in offspring; The attenuated Salmonella recombinant engineered bacteria with sifA gene knockout can inhibit the progression of subcutaneous melanoma xenografts or prolong the survival time of melanoma-bearing mice.

[0026] The fourth aspect of the present application provides the application of attenuated Salmonella recombinant engineered bacteria with sifA gene knockout in the preparation of anti-tumor drugs.

[0027] The fifth aspect of the present application provides the use of a recombinant attenuated Salmonella engineering bacterium with sifA gene knockout in combination with other anti-tumor drugs in the preparation of anti-tumor drugs.

[0028] Furthermore, the tolerance of the transgenic strain is verified by adding a preservative.

[0029] Furthermore, the preservative is potassium sorbate.

[0030] Beneficial effects: Through the sifA gene knockout strategy, the present invention optimizes the tumor targeting, immunocompatibility and therapeutic potential of the strain while reducing toxicity, providing a safer and more efficient engineering solution for bacteria-mediated tumor treatment. The sifA gene knockout VNP20009 attenuated Salmonella strain of the present invention has higher in vivo safety, a higher load in macrophages, and can target tumors and inhibit tumor growth, and is a highly potential chassis strain.

[0031] Compared with the existing invention, the present invention has the following advantages:

[0032] (1) Significantly reduce biological toxicity:

[0033] Compared with the parental strain VNP20009, the engineered bacterium with sifA gene knockout significantly reduces the killing ability against immune cells (such as macrophages), and at the same time its median lethal dose (LD50) for mice is increased by about 9 times or more, greatly improving the in vivo safety. Engineered Salmonella VNP20009 has achieved a series of beneficial effects in solid tumor treatment, but its biological toxicity, especially its toxicity to immune cells, limits the application of this strain in tumor treatment, especially in the application of live anti-tumor drugs combined with synthetic biology. It reduces the distribution of bacteria in the liver and spleen, further reducing the toxicity of non-target organs.

[0034] (2) Enhance tumor targeting and therapeutic effect:

[0035] The present invention retains the ability of the parental strain VNP20009 to specifically colonize the tumor hypoxic region and can be precisely located in the core region of solid tumors. In a melanoma mouse model, it significantly inhibits tumor progression and prolongs the survival time of tumor-bearing mice.

[0036] (3) Improve the immune cell loading capacity:

[0037] Due to the deletion of the sifA gene, the formation of Salmonella-containing vacuoles (SCVs) is blocked, and the bacteria are free in the cytosol, increasing the intracellular load of macrophages on the bacteria, providing an advantage for subsequent immune cell-mediated tumor treatment.

[0038] (4) As an ideal chassis strain for synthetic biology therapy:

[0039] The present invention retains the ability to express exogenous proteins while reducing toxicity to host cells, facilitating the loading of anti-tumor molecules (such as cytokines, toxins, etc.) through genetic engineering, and laying a foundation for the development of combination therapies.

[0040] (5) Reducing the overreaction of the immune system:

[0041] The deletion of sifA in the present invention results in the inability of bacteria to proliferate excessively within macrophages, avoiding excessive damage to immune cells, reducing the inflammatory response, and enhancing treatment tolerance.

[0042] (6) Flexible formulation forms and potential for combination applications:

[0043] The present invention can be formulated into various dosage forms such as oral, injection, intravenous, or spray, and can be used in combination with other anti-tumor drugs to enhance the synergistic therapeutic effect.

[0044] (7) Genetic stability:

[0045] The genotype and phenotype of the sifA gene deletion in the present invention are stably inherited in progeny strains, ensuring the consistency and reliability of the engineered bacteria in applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following described drawings are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0047] Figure 1 Agarose gel electrophoresis diagram of genomic PCR products of the attenuated Salmonella typhimurium recombinant engineered bacteria with sifA gene knockout provided in the present invention and its parent strain VNP20009

[0048] Figure 2 Growth curves of the attenuated Salmonella typhimurium recombinant engineered bacteria with sifA gene knockout provided in the present invention and its parent strain VNP20009 in liquid medium measured at 37°C for 24 hours

[0049] Figure 3 Comparison of the expression abilities of red and green fluorescent proteins of the attenuated Salmonella typhimurium recombinant engineered bacteria with sifA gene knockout provided in the present invention and its parent strain VNP20009 measured at 37°C

[0050] Figure 4 Effects of the attenuated Salmonella typhimurium recombinant engineered bacteria with sifA gene knockout provided in the present invention and its parent strain VNP20009 on the apoptosis of RAW264.7 cells at different invasion ratios

[0051] Figure 5 Comparison of the intracellular loading amounts of the attenuated Salmonella recombinant engineering bacteria with sifA gene knockout provided in the present invention and its parent strain VNP20009 in RAW264.7 live cells

[0052] Figure 6 Survival curves of mice intraperitoneally injected with the attenuated Salmonella recombinant engineering bacteria with sifA gene knockout provided in the present invention and its parent strain VNP20009

[0053] Figure 7 Tumor growth curve graph of melanoma mice after treatment with the attenuated Salmonella recombinant engineering bacteria with sifA gene knockout provided in the present invention

[0054] Figure 8 Graph of the change in body weight of melanoma mice after treatment with the attenuated Salmonella recombinant engineering bacteria with sifA gene knockout provided in the present invention

[0055] Figure 9 Organ index graph of the ratio of the weights of the liver and spleen of melanoma mice to their body weights after treatment with the attenuated Salmonella recombinant engineering bacteria with sifA gene knockout provided in the present invention

[0056] Figure 10 Colony counts in the liver, spleen, and tumor of melanoma mice and the ratios of the colony counts in the tumor to those in the liver and spleen after treatment with the attenuated Salmonella recombinant engineering bacteria with sifA gene knockout provided in the present invention Detailed implementation manners

[0057] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention

[0058] In this application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone. Wherein A and B may be singular or plural. The character " / " generally represents an "or" relationship between the front and rear associated objects

[0059] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following items" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0060] It should be understood that in various embodiments of this application, the magnitudes of the serial numbers of the above - mentioned processes do not imply the sequence of execution. Some or all of the steps can be executed in parallel or sequentially. The execution sequence of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0061] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The singular forms "a", "the", and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0062] The weight of the relevant components mentioned in the specification of the embodiments of this application not only can refer to the specific content of each component, but also can represent the proportional relationship of the weights between the components. Therefore, as long as the content of the relevant components in the specification of the embodiments of this application is scaled up or down proportionally, it is within the scope disclosed in the specification of the embodiments of this application. Specifically, the mass mentioned in the specification of the embodiments of this application can be mass units well - known in the chemical field such as μg, mg, g, kg, etc.

[0063] The terms "first" and "second" are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of this application, the first XX can also be called the second XX, and similarly, the second XX can also be called the first XX. Thus, the features defined with "first" and "second" can explicitly or implicitly include one or more of such features.

[0064] In the first aspect of the embodiments of this application, a recombinant attenuated Salmonella engineering bacterium with sifA gene knockout is provided. The Salmonella recombinant engineering bacterium is a recombinant attenuated Salmonella engineering bacterium with sifA gene knockout derived or genetically modified based on the attenuated Salmonella typhimurium VNP20009.

[0065] In some embodiments, the attenuated Salmonella recombinant engineered bacterium with sifA gene knockout is a derivative engineered strain of engineered Salmonella VNP20009 with sifA gene deletion. The sifA gene of the parental strain VNP20009 is deleted, and this derivative strain can specifically colonize the core region of solid tumors; it has low toxicity to immune cells and high intracellular loading capacity of macrophages; its LD50 in mice is more than 9 times higher than that of its parental strain VNP20009.

[0066] The second aspect of the embodiments of the present application provides an agent of an attenuated Salmonella recombinant engineered bacterium with sifA gene knockout.

[0067] The derivative engineered bacterium of engineered Salmonella VNP20009 with sifA gene deletion can specifically colonize the core region of solid tumors including melanoma, while maintaining low toxicity to immune cells; the complete nucleotide sequence of the genomic potB to downstream gene potD segment is as shown in SEQ NO.2.

[0068] In some embodiments, its active ingredient is at least one of the following (a), (b), (c), and (d):

[0069] (a) Attenuated Salmonella recombinant engineered bacterium cells;

[0070] (b) Ultrasonic disruption precipitate of attenuated Salmonella recombinant engineered bacterium;

[0071] (c) Primary extract of the fermentation broth of the obtained attenuated Salmonella recombinant engineered bacterium culture;

[0072] (d) Ultrasonic lysate supernatant of the obtained attenuated Salmonella recombinant engineered bacterium cells.

[0073] In some embodiments, the preparation of the attenuated Salmonella recombinant engineered bacterium with sifA gene knockout includes oral preparations, injection preparations, intravenous preparations or spray preparations.

[0074] The third aspect of the embodiments of the present application provides a preparation method of an attenuated Salmonella recombinant engineered bacterium with sifA gene knockout, which is characterized by including the following steps:

[0075] (1) Construction of the sifA knockout vector pTAT-sifA-KO plasmid: Design sgRNA, then amplify the upstream and downstream fragments of sifA by PCR, and use the one-step seamless cloning method to clone them into the pTAT vector; after homologous recombination, chemically transform them into Escherichia coli TOP10; after sequencing, the nucleotide sequence of this vector is as shown in SEQ NO.1;

[0076] (2) Electroporate pTAT-sifA-KO into Salmonella typhimurium VNP20009 containing the pCas9 plasmid, culture for gene knockout, and then verify by agarose gel electrophoresis.

[0077] (3) Eliminate the plasmid from the correctly edited strain. Use IPTG induction to eliminate the pTAT-sifA-KO plasmid, and then culture at 42 °C to eliminate the pCas9 plasmid, obtaining a recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout.

[0078] In some embodiments, the genotype and phenotype of sifA gene deletion can be stably inherited in offspring; the recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout can inhibit the progression of subcutaneous melanoma xenografts or extend the survival time of melanoma-bearing mice.

[0079] The fourth aspect of the embodiments of the present application provides an application of a recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout in the preparation of an anti-tumor drug.

[0080] The fifth aspect of the embodiments of the present application provides an application of a recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout combined with other anti-tumor drugs in the preparation of an anti-tumor drug.

[0081] In some embodiments, verify the tolerance of the transgenic strain by adding a preservative.

[0082] In some embodiments, the preservative is potassium sorbate.

[0083] Example 1

[0084] A recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout according to the present invention, wherein the recombinant engineered strain of Salmonella typhimurium is a recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout derived or genetically modified based on attenuated Salmonella typhimurium VNP20009.

[0085] The recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout is a derivative engineered strain of engineered Salmonella typhimurium VNP20009 with sifA gene deletion. The sifA gene of the parental strain VNP20009 is deleted, and this derivative strain can specifically colonize the core region of solid tumors; it has low toxicity to immune cells and high macrophage intracellular loading capacity; its LD50 for mice is more than 9 times higher than that of its parental strain VNP20009.

[0086] Example 2

[0087] A bacterial agent of a recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout according to the present invention.

[0088] The engineered bacterium derived from the engineered Salmonella typhimurium VNP20009 with sifA gene deletion can specifically colonize the core region of solid tumors including melanoma, while maintaining low toxicity to immune cells; the complete nucleotide sequence of the genomic region from potB to the downstream gene potD is shown in SEQ NO.2.

[0089] Its active ingredient is at least one of the following (a), (b), (c), and (d):

[0090] (a) Attenuated Salmonella recombinant engineered bacterium cells;

[0091] (b) Ultrasonic disruption precipitate of attenuated Salmonella recombinant engineered bacterium;

[0092] (c) Primary extract of the fermentation broth of the obtained attenuated Salmonella recombinant engineered bacterium culture;

[0093] (d) Ultrasonic lysate supernatant of the obtained attenuated Salmonella recombinant engineered bacterium cells.

[0094] The preparation of the recombinant engineered bacterium of attenuated Salmonella with sifA gene knockout includes oral preparations, injection preparations, intravenous preparations or spray preparations.

[0095] Example 3

[0096] A method for preparing a recombinant engineered bacterium of attenuated Salmonella with sifA gene knockout according to the present invention includes the following steps:

[0097] (1) Construction of the sifA knockout vector pTAT-sifA-KO plasmid: Design sgRNA, then amplify the upstream and downstream fragments of sifA by PCR, and use one-step seamless cloning into the pTAT vector; after homologous recombination, chemically transform it into Escherichia coli TOP10; after sequencing, the nucleotide sequence of this vector is shown in SEQ NO.1;

[0098] (2) Electroporate pTAT-sifA-KO into Salmonella typhimurium VNP20009 containing the pCas9 plasmid, culture for gene knockout and then verify by agarose gel electrophoresis;

[0099] (3) Eliminate the plasmid of the correctly edited strain, use IPTG induction to eliminate the pTAT-sifA-KO plasmid, and then culture at 42°C to eliminate the pCas9 plasmid to obtain the recombinant engineered bacterium of attenuated Salmonella with sifA gene knockout.

[0100] The genotype and phenotype of the sifA gene deletion can be stably inherited in the offspring; the recombinant engineered bacterium of attenuated Salmonella with sifA gene knockout can inhibit the progression of subcutaneous melanoma xenografts or extend the survival time of melanoma-bearing mice.

[0101] Example 4

[0102] Application of a recombinant attenuated Salmonella engineering bacterium with sifA gene knockout of the present invention in the preparation of anti-tumor drugs.

[0103] Example 5

[0104] Application of a recombinant attenuated Salmonella engineering bacterium with sifA gene knockout of the present invention combined with other anti-tumor drugs in the preparation of anti-tumor drugs. The tolerance of the transgenic strain was verified by adding preservatives. The preservative is potassium sorbate.

[0105] Example 6

[0106] Construction of a recombinant attenuated Salmonella engineering bacterium with sifA gene knockout

[0107] Construction of the knockout vector pTAT-sifA-KO: The sgRNA sequence was obtained by calculation and subcloned into a specific site of the vector pTAT preserved in the laboratory using homologous recombination method; 1500 bp upstream and downstream of the sgRNA were selected as the upstream and downstream homologous arm sequences respectively. The nucleotide sequence of the sgRNA is shown in SEQ NO.3; the nucleotide sequence of the upstream homologous arm sequence of the sifA knockout vector is shown in SEQ NO.4; the nucleotide sequence of the downstream homologous arm sequence of the sifA knockout vector is shown in SEQ NO.5; the nucleotide sequence of the sifA gene knockout detection primer sequence sifA-F is shown in SEQ NO.6; the nucleotide sequence of the sifA gene knockout detection primer sequence sifA-R is shown in SEQ NO.7;

[0108] Using the genome of the parental strain VNP20009 as a template, two target fragments were obtained by PCR and subcloned into specific sites of the vector pTAT to obtain the knockout vector pTAT-sifA-KO. The full vector sequence is shown in SEQ NO.2; the above construction process was completed using a one-step seamless cloning kit and its supporting design software;

[0109] Plasmid transformation: Chemically transform 10 μL of the ligation product into Escherichia coli TOP10 chemically competent cells. The transformation conditions are as follows: Place on ice for 30 minutes, heat shock at 42 °C for 90 seconds, place on ice for 2 minutes, add 900 μL of antibiotic-free LB liquid medium, incubate with shaking at 37 °C for 60 min, centrifuge at 5000 rpm for 3 minutes to remove the supernatant, add 100 μL of antibiotic-free LB liquid medium and mix well, spread on a resistance plate containing 100 μg / mL ampicillin, and incubate overnight in a 37 °C incubator; Pick four monoclonal colonies into 1 mL of liquid LB containing 100 μg / mL ampicillin, incubate with shaking at 37 °C and 220 rpm for 8 hours, obtain the recombinant plasmid using a plasmid miniprep kit, send it for sequencing and detection, and obtain the recombinant plasmid with the correct sequence after comparison.

[0110] Plasmid electroporation: Prepare wild-type VNP20009 (abbreviated as pCas9-VNP) competent cells containing the pCAS9 plasmid: Inoculate the activated pCas9-VNP into 10 mL of kanamycin-resistant LB, culture in a shaker at 30 °C for 1.5 h, add an arabinose inducer with a final concentration of 1% (W / V), continue to culture until the OD600 value reaches 0.6, centrifuge at 5000 rpm and 4 °C for 5 min to collect the bacterial cells, wash the bacterial cells 3 times with 10 mL of 10% glycerol, centrifuge at 5000 rpm and 4 °C for 5 min to collect the bacterial cells, resuspend with 100 μL of 10% glycerol, and dispense 50 μL per tube for electroporation; Electroporate the above recombinant plasmid into pCas9-VNP competent cells by electroporation method: All operations are carried out under sterile conditions. Add 1 μg of the recombinant plasmid to the electroporation competent cells and transfer them to a 2 mm electroporation cuvette. The electroporation conditions are: voltage 1800 - 2500 V, resistance 400 - 500 Ω, capacitance 25 μF, and discharge time 5 ms; Spread the electroporation product on a double-resistant plate containing kanamycin and ampicillin, and incubate overnight in a 30 °C incubator.

[0111] Knockout detection: Pick the above monoclonal colonies, culture them in double-resistant liquid LB containing kanamycin and ampicillin at 30 °C and 220 rpm for 8 h. Then extract the genomic DNA as a template, perform PCR using upstream and downstream detection primers, and detect the band size using 1% agarose gel electrophoresis. Select the colonies with bands conforming to the theoretical size as positive colonies with successful knockout.

[0112] Resistance elimination: The above-mentioned positive colonies were cultured at 30 °C and 220 rpm in kanamycin-resistant LB. When the OD reached about 0.6, an IPTG solution with a final concentration of 1 mg / mL was added, and after overnight culture, streaking was performed on single-resistant plates of kanamycin and ampicillin respectively. If colonies were only formed on the kanamycin plate, it indicated that the pTAT plasmid was successfully eliminated; continue to pick monoclonal colonies on the kanamycin plate and culture them overnight in a kanamycin-free LB medium at 42 °C; streak the bacterial solution on the kanamycin plate, and if no colonies were formed on the kanamycin-resistant plate, it indicated that the resistance elimination was completed, and the strain was preserved.

[0113] Strain verification: Extract the genomic DNA of the strain, perform PCR using upstream and downstream primers, cut the PCR product with agarose gel and send it for sequencing, and compare the results with the genome of wild VNP20009.

[0114] Experimental Example 1

[0115] Test on the growth curve and protein production of the recombinant attenuated Salmonella engineering bacteria with sifA gene knockout at 37 °C for 24 h

[0116] The frozen glycerol bacteria were transferred to fresh medium at a ratio of 1:100 and cultured overnight at 37 °C and 220 rpm. 10 μL of each strain was transferred to 990 μL of fresh LB medium, and mixed well by pipetting. Then, 10 μL was transferred to 990 μL of fresh LB medium again. After mixing, 200 μL per well was transferred to a 96-well plate, and 4 replicates were set for each sample. Using an enzyme-linked immunosorbent assay (ELISA) reader, the growth curve of bacteria was continuously detected for 24 hours. The program settings were: temperature 37 °C, double-track continuous shaking plate, running time 24 h, and detection every 30 min; optical components: top, gain: 100, Read 1: 600, Read 2: 479,520 (EGFP), Read 3: 545,591 (RFP).

[0117] Experimental Example 2

[0118] Determination of apoptosis and intracellular load of RAW264.7 cells by the recombinant attenuated Salmonella engineering bacteria with sifA gene knockout and its parental strain VNP20009 at different invasion ratios

[0119] Cell apoptosis assay: RAW264.7 cells were seeded in 6-well plates at a density of 5×105 cells / well and cultured normally for 12 h before the experiment. The overnight cultured VNP20009 and its derivative engineered strains were centrifuged at 5000 rpm for 5 min at 4 °C, the supernatant was discarded, and the bacterial pellet was resuspended in sterile 1×PBS. The OD values of the bacterial suspensions were adjusted to 1. According to different invasion ratios, the bacterial suspensions were added to the cell samples and co-incubated for 2 h. B16F10 cells were digested with trypsin, centrifuged at 2000 rpm for 5 min at 4 °C, and the cells were collected. The cells were washed once with pre-cooled 1×Binding buffer, 100 μL of 1×Binding buffer containing Annexin V-APC was added, and the cells were gently resuspended by pipetting and incubated in the dark at 4 °C for 30 min. 100 μL of 1×Binding buffer containing PI was added to each sample, gently mixed, and incubated in the dark at 4 °C for 10 min. The apoptosis of the cells was detected by flow cytometry.

[0120] Intracellular load determination: RAW264.7 cells were seeded in 6-well plates at a density of 5×105 cells / well and cultured normally for 12 h before the experiment. The overnight cultured VNP20009 and its derivative engineered strains were centrifuged at 5000 rpm for 5 min at 4 °C, the supernatant was discarded, and the bacterial pellet was resuspended in sterile 1×PBS. The OD values of the bacterial suspensions were adjusted to 1. According to an invasion ratio of 100:1, 125 μL of the bacterial suspension was added to the cell samples and co-incubated for 2 h. The culture medium was discarded, and the cells were washed 3 times with PBS. The cells were then cultured in a medium containing 100 μg / mL gentamicin for 1 h. The culture medium was discarded, and the cell gaps were rinsed with PBS buffer to remove the bacteria that did not invade the cells. 500 μL of trypsin was added to each well to digest the cells, and then 500 μL of serum-containing culture medium was added to terminate the digestion. The digested cells were collected and mixed. A portion of the cell suspension was stained with trypan blue, and the number of live cells was recorded using a cell counter. A small amount of TritonX-100 was added to the remaining cell suspension, and the cells were lysed by pipetting. After dilution, the cell suspension was plated to calculate the number of intracellular bacteria. Finally, the ratio of the total number of intracellular bacteria to the total number of live cells was used to characterize the average number of intracellular bacteria.

[0121] Experimental Example 3

[0122] Determination of survival curve and LD50 by intraperitoneal injection of a derivative engineered strain (hereinafter referred to as dsifA) of the sifA gene-deleted engineered Salmonella typhimurium VNP20009 in mice

[0123] LD50 pre-experiment: Seven-week-old female C57BL / 6 mice were normally fed for 5 days and then inoculated with bacteria at the set concentrations by intraperitoneal injection. The mice were continuously observed for 14 days, and the number of deaths was recorded to obtain the 0% and 100% death doses of each strain.

[0124] LD50 determination experiment: Ninety healthy mice were randomly divided into three groups: the PBS group (10 mice), the VNP20009 group (40 mice), and the dsifA group (40 mice). Four dose groups were set for each strain, with an equal number of male and female mice, 10 mice per group. After normal feeding for five days, the doses were designed according to the results of the preliminary experiment (gradually increasing or decreasing in geometric progression, with the ratio of adjacent two doses being 1:0.6 - 0.93). The mice were injected intraperitoneally, and the death situation and general signs of the mice were observed continuously for 14 days. The LD50 of different strains was calculated by the Bliss method.

[0125] Experimental Example 4

[0126] Determination of tumor growth curve and body weight change curve of melanoma mice treated with the recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout (hereinafter referred to as dsifA)

[0127] Establishment of subcutaneous melanoma model in mice: B16F10 cells were subcutaneously injected into the axillary region of mice. When the tumor volume grew to 90 - 100 mm3, the mice were grouped. The tumor-bearing mice were randomly divided into four groups and intraperitoneally injected with 0.1 ml of PBS, VNP, or dsifA. The dose of the VNP group was 1×10^6 CFU / mouse, and two dose groups were set for dsifA, which were 1×10^6 CFU / mouse and 9×10^6 CFU / mouse, respectively.

[0128] Bacterial treatment: The activated VNP and the engineered strain were cultured until the OD600 reached 0.6 - 0.8, then taken out, centrifuged at 5000 rpm / min for 4 min to collect the bacteria, washed three times with PBS buffer and resuspended. The OD value of the bacterial suspension was adjusted to 1, and then adjusted to the appropriate number of bacteria according to the experimental requirements.

[0129] Data recording: The body weight of the mice was measured every other day, and the length and width of the tumors of the mice were measured using a vernier caliper. At the same time, the status of each mouse was observed and recorded. The experiment was ended when the tumor volume of the mice in the PBS group reached 1500 mm3.

[0130] Experimental Example 5

[0131] Determination of liver and spleen organ indices and bacterial distribution in some organs of melanoma mice after treatment with the recombinant engineered strain of attenuated Salmonella typhimurium with sifA gene knockout (hereinafter referred to as dsifA)

[0132] Establishment of subcutaneous melanoma model in mice: B16F10 cells were subcutaneously injected into the axillary region of mice. When the tumor volume grew to 90 - 100 mm3, the mice were grouped. The tumor-bearing mice were randomly divided into four groups and intraperitoneally injected with 0.1 ml of PBS, VNP, or dsifA. The dose of the VNP group was 1×10^6 CFU / mouse, and two dose groups were set for dsifA, which were 1×10^6 CFU / mouse and 9×10^6 CFU / mouse, respectively.

[0133] Bacterial treatment: The activated VNP and the genetically modified strain were taken out when the OD600 reached 0.6 - 0.8, centrifuged at 5000 rpm / min for 4 min to collect the bacteria, washed three times with PBS buffer and then resuspended. The OD value of the bacterial solution was adjusted to 1, and then adjusted to the appropriate number of bacteria according to the experimental requirements.

[0134] Sampling: On the fourth day after inoculating the bacteria, the mice were sacrificed, and the tumors, livers, and spleens were dissected with surgical scissors and forceps. After taking pictures, they were weighed using an analytical balance and recorded. Part of the tumor, liver, and spleen were cut and weighed, and then homogenized and ground. After gradient dilution, they were spread on LB plates for counting.

[0135] In summary, the present invention uses the attenuated Salmonella typhimurium VNP20009 as the parental strain, and combines gene editing technology to construct a derivative engineering bacterium of Salmonella VNP20009 with lower toxicity to immune cells. It can colonize in the core area of solid tumors. At the same time, the sifA gene of this strain is knocked out, resulting in a weakened toxicity to immune cells. Although its therapeutic effect shows a downward trend compared with VNP20009, its safety has been greatly improved. When injecting an engineering bacterium preparation with a dose similar to the therapeutic effect of VNP20009, the weight loss of the mice caused by it is lower than that of the VNP20009 preparation. At the same time, the intracellular loading amount of immune cells of this strain is much higher than that of VNP20009, and its toxicity to immune cells RAW264.7 is much lower than that of VNP20009. Therefore, this strain has the potential to become a good synthetic biology drug delivery platform, creating an ideal chassis strain for subsequent bacterial therapies combining synthetic biology.

[0136] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification and their equivalents.

Claims

1. A recombinant engineered attenuated Salmonella with sifA gene knocked out, characterized in that: The attenuated Salmonella recombinant engineering bacterium is an attenuated Salmonella recombinant engineering bacterium with sifA gene knocked out, which is derived from or genetically modified attenuated Salmonella typhimurium VNP20009.

2. The attenuated Salmonella recombinant engineered bacterium with sifA gene knockout according to claim 1, characterized in that: The attenuated Salmonella recombinant engineered bacteria with sifA gene knockout are derivative engineered strains of the engineered Salmonella VNP20009 with sifA gene deletion. The derivative strain can specifically colonize the core area of ​​solid tumors; it has lower toxicity to immune cells and higher intracellular loading capacity of macrophages; its LD50 for mice is more than 9 times higher than that of its parent strain VNP20009.

3. The attenuated Salmonella recombinant engineered bacterial agent with sifA gene knocked out according to claim 1.

4. The attenuated Salmonella recombinant engineered bacterial agent with sifA gene knockout according to claim 3, wherein the active ingredient is at least one of the following (a), (b), (c), and (d): (a) the attenuated Salmonella recombinant bacterial cell according to claim 1; (b) ultrasonic disruption precipitation of the attenuated Salmonella recombinant engineering bacteria according to claim 1; (c) a primary extract of the fermentation broth of the attenuated Salmonella recombinant engineering bacterial culture obtained according to claim 1; (d) The supernatant of ultrasonic lysis of the attenuated Salmonella recombinant bacterial cells obtained according to claim 1.

5. The attenuated Salmonella recombinant engineered bacterial agent with sifA gene knockout according to claim 4, characterized in that: The preparations of the attenuated Salmonella recombinant engineered bacteria with sifA gene knocked out include oral preparations, injection preparations, intravenous preparations or spray preparations.

6. The method for preparing the attenuated Salmonella recombinant engineering bacteria with sifA gene knocked out according to claim 1, characterized in that The steps include: (1) Construction of sifA knockout vector pTAT-sifA-KO plasmid: design sgRNA, then use PCR to amplify the upstream and downstream fragments of sifA, and use a one-step method to seamlessly clone into the pTAT vector; after homologous recombination, chemically transform into Escherichia coli TOP10; after sequencing, the nucleotide sequence of the vector is shown in SEQ NO.1; (2) pTAT-sifA-KO was transformed into Salmonella VNP20009 containing the pCas9 plasmid by electroporation, and the gene knockout was cultured and then verified by agarose gel electrophoresis; (3) The correctly edited strain was subjected to plasmid elimination, and IPTG induction was used to eliminate the pTAT-sifA-KO plasmid. The pCas9 plasmid was then eliminated by culturing at 42°C to obtain a recombinant engineered Salmonella strain with sifA gene knockout.

7. The method for preparing the attenuated Salmonella recombinant engineering bacteria with sifA gene knockout according to claim 6, characterized in that: The genotype and phenotype of sifA gene deletion are stably inherited in the offspring; the attenuated Salmonella recombinant engineered bacteria with sifA gene knockout can inhibit the progression of subcutaneous melanoma transplanted tumors or prolong the survival time of melanoma-bearing mice.

8. Use of the attenuated Salmonella recombinant engineering bacteria with sifA gene knockout according to claim 1 in combination with other anti-tumor drugs in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tolerance of the transgenic strains was verified by adding preservatives.

10. The use according to claim 8, characterized in that: The preservative is potassium sorbate.

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