KR20210123556A - Anticancer Salmonella Inducing Immune Response by Control of Filamentous Growth and Anticancer Composition Comprising the Salmonella - Google Patents

Abstract

The present invention relates to a novel Salmonella strain that can be effectively used for anticancer treatment by being controlled for filament growth after being targeted to a tumor site, has resistance to an attack by immune cells and has excellent efficacy in inducing an immune response, and an anticancer composition including the same, and more specifically, to an attenuated anticancer Salmonella strain that is transformed by a gene structure including a filament growth inducing gene operably linked to an inducible promoter, and an anticancer composition including the same.

Description

Anticancer Salmonella Inducing Immune Response by Control of Filamentous Growth and Anticancer Composition Comprising the Salmonella

The present invention relates to a novel Salmonella strain that can be effectively used for anticancer treatment because it has resistance to attack by immune cells and has excellent efficacy in inducing immune response by being controlled to grow filaments after being targeted to a tumor site, and an anticancer composition comprising the same will be.

Cancer is a life-threatening disease by which cells do not stop proliferating and invade surrounding tissues and destroy normal cells. As the control ability of the human body decreases due to aging, it becomes more vulnerable to cancer. In an aging society, cancer has been the number one cause of death for 10 years since 2007, more than double the death rate from heart disease, the second largest. .

The most effective response system for the treatment of disease is to strengthen the immune system, but since cancer cells are not foreign invaders, they are avoided without triggering the body's immune response. Certain viruses or bacteria infect cancer cells more than normal cells, and the infected bacteria can become targets for immune cells to attack. Accordingly, anticancer treatment methods have been proposed that allow the body to fight cancer cells by intentionally infecting a specific virus or bacteria to stimulate the body's immune response. Infectious bacteria such as Shigella, Vibrio cholera, and pathogenic E. coli only infiltrate the cells of the intestinal tract, but do not reach the liver and spleen, which are important organs for generating an immune response. In contrast, Salmonella can penetrate the spleen and liver through the lymph nodes and stimulate a systemic immune response. Specifically, Leschner et al. (J. Mol. Med. 2010, 88, 763-773) were intravenously infected with fluorescence Salmonella in mice having CT26 tumors, and then traced the infection route over time. . As a result, it was shown that the whole body was infected through mouse blood immediately after infection, and Salmonella bacteria were accumulated in the spleen and liver 20 minutes after infection, and it was observed that Salmonella bacteria were intensively accumulated only in the tumor tissue after 24 hours.

However, Salmonella is a representative bacteria that causes food poisoning and can be life-threatening by causing sepsis by infection, so it is too pathogenic to be used directly for cancer treatment. A research team at Yale University in the United States announced that by genetic manipulation of Salmonella, it can be attenuated by removing only toxicity while maintaining its ability to attack tumors, and that it can suppress tumors by inducing immune stimulation through injection of the attenuated Salmonella. However, the attenuated Salmonella has low viability and reduced immunity, and there is a risk that the attenuated strain is converted to wild-type Salmonella due to mutation to cause sepsis. In addition, due to the lack of R&D funds, anticancer treatment using attenuated salmonella is mostly stopped or withheld in the first stage of the clinical process.

The induction of sepsis, which may accompany cancer treatment using bacteria such as Salmonella, is a very important problem to be overcome, and in addition, stimulation of the immune response must also be actively performed.

Accordingly, studies were conducted to develop strains capable of additionally expressing or suppressing substances useful for anticancer treatment along with attenuation and immune activation, and to use them for anticancer treatment. Registered Patent No. 10-0852687 discloses a Salmonella strain expressing tumor necrosis factor alpha transduced with a tumor necrosis factor alpha protein vector into an attenuated Salmonella strain and a composition for anticancer treatment containing the same, and registered patent No. 10 No.-1750007 published a solid cancer treatment using an attenuated Salmonella strain in which L-asparaginase can be selectively expressed at tumor sites. However, although the above Salmonella strains additionally supplement their anticancer activity by the expression of an anticancer substance, the low viability of attenuated Salmonella and the risk that the attenuated strain is converted to wild-type Salmonella to cause sepsis still has.

On the other hand, Lim et al. (PLos One, 2012; 7(9):e45236) found that the ninth amino acid of the oriC-binding protein of E. coli, Cnu protein (OriC-binding nucleotide-associated protein), is converted from lysine (K) to glutamic acid (G). ), it has been reported that E. coli grows filamentous in a temperature-dependent manner, and according to Prashar et al. (J. Cell Biol. 2013 Vol. 203 No. 6 1081-1097), the filamentous phase of bacteria inhibits phagocytosis, It has been reported that it can withstand the attack of immune cells for a longer period of time. However, it has not been reported that these properties can be usefully used for anticancer treatment.

Registered Patent No. 10-0852687 Registered Patent No. 10-1750007

Leschner et al., J. Mol. Med. 2010, 88, 763-773. Lim et al., PLos One, 2012; 7(9):e45236. Prashar et al., J. Cell Biol. Vol. 203 No. 6 1081-1097.

An object of the present invention is to provide a novel anti-cancer Salmonella with excellent immunity-inducing effect and improved safety while resisting the attack of immune cells longer in order to solve the problems of the prior art attenuated Salmonella during anticancer treatment.

Another object of the present invention is to provide an anticancer composition using the strain.

The present invention for achieving the above object relates to an anticancer attenuated Salmonella strain characterized in that it is transformed by a gene construct comprising a filamentous growth inducing gene operably linked to an inducible promoter.

Salmonella strains are attracting attention as very promising candidates for anticancer therapy because they suppress tumors by specifically inhibiting immunostimulation at the tumor site after being targeted to tumor cells. However, when attenuated in order to remove the toxicity of Salmonella, there is a problem in that the anticancer efficacy is lowered and safety is also not guaranteed because the viability and immunity-inducing ability in the body are reduced.

In the present invention, by inducing Salmonella to grow filaments, it inhibits phagocytosis by macrophages and survives for a long time in the body, and it is characterized in that it has excellent anticancer efficacy even when it is attenuated by stimulating the immune response. In addition, when filaments grow after being targeted to the tumor site, the filamentous strain exhibits the effect of being immobilized at the tumor site because its mobility is reduced. Therefore, the immune response can be stimulated more specifically at the tumor site, and safety is also improved because it does not migrate to other cells.

In the present invention, the filament growth inducing gene may be a CnuK9E gene as in the following Examples. However, if it can induce filament growth in the Salmonella strain, it is of course not limited thereto.

As used herein, the term "inducible promoter" refers to a promoter capable of switching the operation of a gene linked by an inducer. Examples of inducible promoters include tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, pBAD promoter, Tet promoter, trc promoter , pepT promoter, sulA promoter, pol 11 (dinA) promoter, ruv promoter, uvrA promoter, uvrB promoter, uvrD promoter, umuDC promoter, lexA promoter, cea promoter, caa promoter, recN promoter, pagC promoter, hip promoter, ansB promoter and each may be selected from the group consisting of the pflE promoter.

As used herein, the term "attenuation" refers to artificially weakening the toxicity of a living pathogen, mutating genes involved in essential metabolism of the pathogen to prevent disease in the body, and to induce immunity by stimulating only the immune system. Genes that cause attenuation of Salmonella are well known in the art and include aroA, aroC, aroD, aroE, Rpur, htrA, ompR, ompF, ompC, galE, cya, crp, cyp, phoP, phoQ, rfaY, dksA, hupA, selected from the group consisting of sipC, clpB, clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, relA and spoT loss of function of one or more genes. It is more preferable to lose the function of two or more of the above genes in order to prevent reduction to the wild type and further attenuate toxicity upon in vivo application.

In the following examples, when the tetRa promoter, a type of Tet promoter, was used as an inducible promoter, the loss of aroA function was induced to grow the longest in the aroA- strain and aroA galE- strain, and in some attenuated strains, it was induced by doxycycline. It could not be confirmed that filament growth was induced. However, the above result does not mean that the attenuated strain cannot be used in the present invention, and it may still be useful when other inducible promoters are used, so it is not excluded.

On the other hand, since the gene artificially inserted by transformation is not essential for the survival of bacteria, the ability to induce filament growth may be lost due to the removal of the plasmid in the in vivo proliferation process. Accordingly, in order to stably maintain the plasmid, the attenuated Salmonella strain preferably has a glmS gene deleted, and the plasmid contains the glmS gene. Due to this structure, since the glmS gene expressed by the plasmid is essential for the survival of the Salmonella strain, the plasmid can be stably maintained in vivo.

As a result of the experiment using the tetRA promoter in the following examples, the strain with the best filament growth efficacy was named Cnu-K9E glmS / aroA galE glmS Salmonella, and deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on March 20, 2020. (Accession No. KCTC18810P).

The present invention also relates to an anti-cancer composition containing the anti-cancer attenuated Salmonella strain as an active ingredient. When the present composition is used, after administering the anticancer attenuated Salmonella strain, when the strain is targeted to cancer tissue, an agent for inducing the filament growth of the strain by inducing the operation of the inducible promoter. can

The composition of the present invention may be prepared by a method known in the pharmaceutical field in order to be used as an anticancer drug, by itself or by mixing with a pharmaceutically acceptable carrier, excipient, diluent, etc. can be used

The composition of the present invention may be formulated and used as a formulation for oral or parenteral administration. The dosage of the active ingredient according to the present invention may be appropriately selected according to the absorption of the active ingredient in the body, the form of the preparation, the age, sex and condition of the patient, the severity of symptoms, etc., and the administration may be administered once a day. , may be administered in several divided doses. A typical dosage is 0.001 mg/kg·day to 10 g/kg·day.

In addition, the anticancer attenuated Salmonella strain of the present invention can be used as an attenuated strain for transformation. That is, it can be used by additionally transforming to secrete a substance having an anticancer effect by being targeted to a tumor site, or to increase or suppress the expression of a substance that increases anticancer efficacy. To this end, the present invention provides a recombinant attenuated Salmonella strain transduced with a gene construct comprising a gene encoding a foreign protein operably linked to the anticancer attenuated Salmonella strain or a gene for inhibiting or promoting the expression of a specific gene. . The gene construct may be contained in a vector or inserted into the chromosome of the anticancer attenuated Salmonella strain, and transduction may be performed using any method known in the art.

The exogenous protein may be a protein having anticancer efficacy by itself or a metabolite having an anticancer effect, or may be a diagnostic marker protein. The target gene is a gene directly or indirectly involved in the growth of cancer cells, and may be one capable of inhibiting the growth of cancer cells by suppressing or promoting their expression.

As described above, the Salmonella strain according to the present invention has strong resistance to attack by immune cells by inducing the growth of filaments by the operation of an inducer after being targeted to cancer, has excellent effect of attracting immune cells, and can be transferred to other tissues. Its mobility is reduced and safety is also excellent, so it can be more usefully used for anticancer treatment.

In addition, the Salmonella strain of the present invention can be further transformed to enhance or suppress the expression of substances helpful for anticancer treatment at the tumor site to further enhance the efficacy of anticancer treatment.

1 is a schematic diagram of a Salmonella strain according to an embodiment of the present invention.
Figure 2 is a cleavage map of the plasmid into which the CnuK9E gene used in an embodiment of the present invention is inserted.
Figure 3 is a photomicrograph showing the filament growth induction of Salmonella by the expression of CnuK9E gene.
Figure 4 is a micrograph showing the filament growth induction of pCnuK9E / KST0650 strain according to an embodiment of the present invention.
Figure 5 is a micrograph showing the filament growth induction of pCnuK9E / ppGpp- strain according to an embodiment of the present invention.
Figure 6 is a micrograph showing the filament growth induction of pCnuK9E / aroA- strain according to an embodiment of the present invention.
7 is a micrograph showing the filament growth induction of Cnu-K9E glmS / aroA galE glmS Salmonella strain according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, these embodiments are merely examples for easily explaining the content and scope of the technical idea of the present invention, and thereby the technical scope of the present invention is not limited or changed. It will be natural for those skilled in the art that various modifications and changes can be made within the scope of the technical spirit of the present invention based on these examples.

[Example]

Pre-experiment: Filament growth induction assay of Salmonella strain by transformation of CnuK9E gene

First, it was confirmed by transformation whether the CnuK9E gene, which is known to induce filament growth in a temperature-dependent manner in E. coli, can induce filament growth in Salmonella strains as well.

The plasmid PHL887 into which the CnuK9E gene was inserted was received from the laboratory of Professor Heon-Man Lim of Chungnam National University, and wild-type Salmonella LT2 was transformed using the plasmid. 2 is a cleavage map of the corresponding plasmid. As shown in FIG. 3, transformed Salmonella was confirmed that filament growth was induced by IPTG, and filamentous growth of Salmonella strains could be induced by expression of the CnuK9E gene.

Accordingly, a Salmonella strain capable of inducing the filament growth of Salmonella in an actual living body was prepared.

Example 1: Preparation of Salmonella strains with controlled filament growth

As a specific example, using the tetRA promoter and the CnuK9E gene as an inducible promoter and KST0650 Salmonella as the attenuated Salmonella, a Salmonella strain in which filament growth is regulated was prepared.

1) Construction of tetRA pUC19 plasmid

First, using the genomic DNA of Salmonella strain TH3847 containing the tetRA gene (obtained from Professor Im Heon-man, Chungnam National University) as a template, and using the primers in Table 1 below, a PCR product in which tetRA is inserted was obtained by PCR. 10x buffer 5μl, 5x Q solution 10 μl, 2 mM dNTPs 7.5 μl, 20 μM TetRA-hindIII-for primer 2.5 μl, 20 μM TetRA-bamH1-re 2.5 μl, Taq polymerase 1 μl, 10 ng/μl genomic DNA 2 μl And 19.5 μl of water was mixed and PCR was performed using the Qiagen system. PCR conditions were repeated for 5 minutes at 95°C, 30 cycles of 94°C for 30 seconds, 49°C for 30 seconds, 72°C for 2 minutes and 72°C for 10 minutes.

After the product obtained by the PCR was purified by PCR purification kit (Qiagen Co., Ltd.), 1 ug of PCR fragment was reacted at 37° C. for 2 hours using 10 U each of HindIII (Takara Co., Ltd.) and BamHI (Takara Co., Ltd.). did it The reaction product was purified with a PCR purification kit (Qiagen Co., Ltd.) to prepare tetRA insert DNA with a size of 1.9 kb.

pUC19 DNA was reacted with HindIII and BamHI at 10 U and 37° C. for 2 hours, respectively, and electrophoresed. The pUC19 vector was obtained by extracting DNA from the gel using a gel extraction kit (Qiagen, Ltd.).

tetRA insert and pUC19 vector were ligated (Invitron) at 25°C for 30 minutes, and DH5a competent cells were transformed. Transformed cells were spread on LB amp solid medium and cultured at 37° C. to select antibiotic-resistant colonies. Six candidates of the selected colonies were selected and reacted with HindIII 10 U and BamHI 10 U at 37° C. for 1 hour, and the generation of a 1.9 kb band was confirmed through electrophoresis. By requesting Cosmogenetech to analyze the candidate's nucleotide sequence, insertion of the tetRA gene was confirmed, and the tetRA pUC19 plasmid was established.

2) Introduction of CnuK9E gene into tetRA pUC19 plasmid

PCR was performed using the primers of Table 2 below and PrimeSTAR Max DNA Polymerase 2X master mix (Takara Co., Ltd.) using the CnuK9E gene inserted plasmid PHL887 as a template. PCR was performed under the conditions of 98°C for 3 minutes, 35 cycles of 98°C for 10 seconds, 55°C for 5 seconds, 72°C for 5 seconds, and 72°C for 3 minutes. After the PCR product was reacted with 10U of each of BamHI (Takara Co., Ltd.) and SacI (Takara Co., Ltd.) at 37°C for 1 hour, electrophoresis was carried out as in 1), DNA was extracted from the gel, and Cnu-K9E Insert with a size of 300 b was obtained. produced.

The tetRA pUC19 plasmid prepared in 1) was BamHI (Takara Co., Ltd.) and SacI (Takara Co., Ltd.) each were reacted at 10U and 37°C for 2 hours, followed by electrophoresis and DNA extraction from gel to prepare a tetRA vector.

After CnuK9E insert and tetRA vector were ligated (Invitron) at 25°C for 30 minutes, DH5a competent cells were transformed. Transformed cells were spread on LB amp solid medium and cultured at 37° C. to select antibiotic-resistant colonies. Six candidates of the selected colonies were selected and reacted with 10U of each of BamHI (Takara Co., Ltd.) and SacI (Takara Co., Ltd.) at 37° C. for 1 hour, and then the generation of 300 b band was confirmed through electrophoresis. By requesting Cosmogenetech to analyze the candidate's nucleotide sequence, the insertion of the CnuK9E gene was confirmed and established as a pCnuK9E plasmid.

3) Confirmation of transformation and filament growth induction of the attenuated Salmonella strain

The attenuated Salmonella strain was transformed using the pCnuK9E plasmid prepared in 2), and it was confirmed whether filament growth was induced. Attenuated Salmonella strains include ppGpp- (sold from Chonnam National University Professor Jaeho Jeong), araA-, Hns-, rfaH-, ompR-, cyaA-, crp- (above, sold from Hannam University Professor Insu Lee), aroA galE-, galE flhDC -(Self-manufactured) strain and KST0650 Salmonella strain (sold by Advanced Radiation Research Institute, Korea Atomic Energy Research Institute) were targeted.

In order to further attenuate the toxicity of the araA- strain by mutation, the aroA galE- strain was self-produced. The araA galE- strain lost the functions of the aroA and galE genes at the same time, and was prepared by the following method. P22 transduction was performed to introduce the galE- Salmonella strain into the araA- Salmonella strain. ^{2 ml of P22 broth (0.2% glucose, 1% EX50 salts, P22 stock ~10 11} /ml) was added to 0.2 ml of the galE- Salmonella strain (sold from Professor Im Heon-man, Chungnam National University) cultured with shaking at 37°C for 16 hours, and then 37 Incubated at ℃ for 9 hours. The supernatant was transferred to a new tube by centrifugation, and then treated with chloroform to prepare P22 lysate containing the galE- mutation. P22 lysate for 37 hours with shaking at 16 ℃ cultured aroA- mutant 0.1 ml and 1: 10 in PBS solution after 1 hour reaction at room temperature (~ 25 ℃) were mixed in a ratio of 1: ^0, 10 ^-1, 10 ^-2 Alternatively, it ^{was diluted at a ratio of 10 -3} and spread on an LB tetracycline plate to select a strain having antibiotic resistance. The selected strains were confirmed by sequencing at Cosmogenetech.

A single colon of each transformed strain was inoculated into LB amp broth, and then cultured with shaking at 37°C for 12 to 16 hours. _{After that, it was diluted to an OD 600} of 0.05 in fresh LB amp liquid medium and cultured with shaking at 37°C for 2 hours. When the OD ₆₀₀ reached 0.4 to 0.6 by shaking culture, doxycycline was treated at a concentration of 0, 20, or 200 ng/ml, and then cultured with shaking at 37° C. for 3 hours and observed under a microscope. 4 to 6 are photomicrographs showing experimental results for KST0650, ppGpp-, and aroA- attenuated strains, respectively. In the figure, the red scale bar indicates 20 μm corresponding to the diameter of macrophages.

In FIG. 4 , it was confirmed that, in the KST0650 strain, after transformation (pCnuK9E/KST0650), filament growth was induced by treatment with doxycycline, and the length of the filament phase was increased depending on the concentration of doxycycline. When 200 ng/ml of doxycycline was treated, the length of the strain was about 20 μm. In FIG. 5 , the ppGpp- strain grew to about 40 μm, slightly longer than pCnuK9E/KST0650, when treated with 200 ng/ml doxycycline after transformation (pCnuK9E/ppGpp-). 6 , the aroA- strain grew to about 40-100 μm after transformation (pCnuK9E/aroA-) when treated with 200 ng/ml doxycycline. The aroA galE- strain exhibited the same filament growth effect as the araA- strain upon transformation.

On the other hand, Hns-, rfaH-, ompR-, cyaA-, crp-, galE flhDC- among the attenuated strains could not confirm the filament growth induction by doxycycline after transformation.

4) Stabilization of plasmid by introduction of glmS gene

In order for the transformed strain to stably maintain the plasmid in vivo, glmS was knocked down in the attenuated strain, and the glmS gene was inserted into the plasmid to prepare a Salmonella strain.

Construction of the araA galE glmS-strain

The glmS- strain was introduced into the aroA glaE- strain prepared in 3) by P22 transduction. ^{For this, 2 ml of P22 broth (0.2% glucose, 1% EX50 salts, P22 stock ~10 11} /ml) was added to 0.2 ml of glmS-Salmonella strain (sold from Professor Jaeho Jeong, Chonnam National University), which was cultured with shaking at 37℃ for 16 hours. Incubated at 37°C for 9 hours. The supernatant was transferred to a new tube by centrifugation, and then treated with chloroform to prepare P22 lysate containing the glmS- mutation. After mixing 0.1 ml of aroA galE- strain cultured with shaking at 37°C for 16 hours at a ratio of 1:1, react at room temperature (~25°C) for 1 hour, and then in PBS solution 100 ⁰ , 10 ^-1 , 10 ^-2 or 10 ^The strains having antibiotic resistance were selected by dilution at a ratio of -3 and smeared on an LB tetracycline plate. The selected strains were confirmed by sequencing at Cosmogenetech.

Construction of Cnu-K9E glmS plasmid

After reacting the pCnuK9E plasmid prepared in 2) with 10U of aatII (Takara Co., Ltd.) at 37°C for 2 hours, electrophoresis was performed and DNA was extracted from the gel to prepare a pCnuK9E aatII vector.

Using the chromosomal DNA of wild-type Salmonella LT2 as a template, the glmS-aat-F and glmS-aat-R primers in Table 3 below and PrimeSTAR Max DNA Polymerase 2X master mix (Takara Co., Ltd.) were used at 98°C for 3 minutes, 35 times at 98°C 10 PCR was performed under conditions of a cycle of sec - 55 °C 5 sec - 72 °C 2 min, 72 °C 3 min. After electrophoresis from the PCR product, glmS inset was prepared through gel extraction.

The pCnuK9E aatII vector and glmS insert were reacted for 2 minutes at room temperature (25° C.) using 0.6 U of T4 DNA polymerase (NEB). Thereafter, the reaction was performed in an ice water bath for an additional 10 minutes, and the cells were transformed into DH5a competent cells and then plated on LB amp solid medium. The colonies with antibiotic resistance were selected by culturing at 37°C, and 6 candidates of the selected colonies were requested to Cosmogenetech and base analysis was performed to confirm the insertion of the glmS gene. The constructed pCnuK9E glmS plasmid was named Cnu-K9E glmS plasmid.

Confirmation of filament growth induction

The aroA galE glmS- strain was transformed with the Cnu-K9E glmS plasmid, and it was confirmed whether filament growth was induced by doxycycline by the method described in 3). 7 is a photomicrograph of a transformed strain upon treatment with doxycycline at a concentration of 200 ng/ml, and it was confirmed that filament growth was induced by doxycycline. Its length was about 100 μm.

The transformed strain was named Cnu-K9E glmS/ aroA galE glmS Salmonella, and was deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on March 20, 2020 (Accession No. KCTC18810P).

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Claims (8)

An anticancer attenuated Salmonella strain, characterized in that it is transformed by a gene construct comprising a filament growth inducing gene operably linked to an inducible promoter.
The method of claim 1,
The filament growth inducing gene is an anticancer attenuated Salmonella strain, characterized in that the CnuK9E gene.
The method of claim 1,
The inducible promoter is tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, pBAD promoter, Tet promoter, trc promoter , pepT promoter, sulA promoter, pol 11 (dinA) promoter, ruv promoter, uvrA promoter, uvrB promoter, uvrD promoter, umuDC promoter, lexA promoter, cea promoter, caa promoter, recN promoter, pagC promoter, hip promoter, ansB promoter and An anticancer attenuated Salmonella strain, characterized in that it is selected from the group consisting of the pflE promoter.
The method of claim 1,
The attenuation causing genes are aroA, aroC, aroD, aroE, Rpur, htrA, ompR, ompF, ompC, galE, cya, crp, cyp, phoP, phoQ, rfaY, dksA, hupA, sipC, clpB, clpP, clpX, loss of function of one or more genes selected from the group consisting of pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, relA and spoT Anti-cancer attenuated Salmonella strain, characterized in that inducing.
5. The method according to any one of claims 1 to 4,
The attenuated Salmonella strain has a glmS gene deleted,
The plasmid is an anticancer attenuated Salmonella strain, characterized in that it contains the glmS gene.
6. The method of claim 5,
The strain is an anticancer attenuated Salmonella strain, characterized in that the accession number KCTC18810P.
An anticancer composition comprising the anticancer attenuated Salmonella strain of any one of claims 1 to 5 as an active ingredient.
6. Recombinant attenuation transduced with a gene construct comprising a gene encoding a foreign protein operably linked to the anticancer attenuated Salmonella strain of any one of claims 1 to 5 or a gene for inhibiting or promoting expression of a target gene. Salmonella strain.

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