KR20230064561A - Anticancer Microbials with Dual Secreting System - Google Patents

Abstract

The present invention relates to an anti-cancer strain and an anti-cancer composition including an anti-cancer as an active ingredient capable of simultaneously secreting proteins expressed by two different genes by including a dual secreting system operated with different mechanisms. The anti-cancer strain is transformed by a gene structure (A) including a gene encoding ClyA protein which is operably connected to a first inducible promoter; and a gene structure (B) including a gene encoding GM-CSF protein connected to an anti-sigma factor flaM gene which is operably connected to a second inducible promoter. The anti-cancer strain mounts the dual secreting system. In addition, the present invention relates to the anti-cancer composition containing the anti-cancer strain as the active ingredient.

Description

Anticancer Microbials with Dual Secreting System}

An object of the present invention is to provide an anticancer strain capable of simultaneously secreting proteins expressed by two different genes by mounting a dual secretion system operating in different mechanisms and an anticancer composition containing the same as an active ingredient.

BACKGROUND OF THE INVENTION Cancer is a life-threatening disease in which the proliferative activity of cells does not stop, penetrates into surrounding tissues and destroys normal cells. As the human body's controllability decreases due to aging, it becomes vulnerable to cancer. In an aging society, cancer has been the undisputed number one cause of death since 2007, surpassing twice the death rate from heart disease, which is the second highest.

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 do not trigger the body's immune response and avoid it. Certain viruses or bacteria infect cancer cells more than normal cells, and the infected bacteria can become targets of attack by immune cells. Accordingly, anti-cancer treatment methods have been suggested in which a specific virus or bacterium is deliberately infected to stimulate the body's immune response to fight cancer cells.

Leschner et al. (J. Mol. Med. 2010, 88, 763-773) infected mice with CT26 tumors with fluorescent Salmonella by intravenous injection, and followed the route of infection over time. As a result, it was shown that the whole body was infected through the blood of the mouse immediately after infection, Salmonella bacteria accumulated in the spleen and liver 20 minutes after infection, and Salmonella bacteria accumulated intensively only in tumor tissues after 24 hours. This suggests that it can be effectively used for anti-cancer immunotherapy by targeting cancer and activating immunity. However, since Salmonella is a representative bacterium that causes food poisoning, it can cause sepsis by infection and threaten life, so its pathogenicity is too strong to be used directly for cancer treatment. A research team at Yale University in the United States announced that genetic manipulation of Salmonella can attenuate it by removing toxicity while maintaining its tumor-attacking properties, and suppressing tumors by inducing immune stimulation through injection of the attenuated Salmonella. Accordingly, studies on strains transformed to express substances having anticancer properties in attenuated Salmonella are being actively conducted. However, attenuated Salmonella has low survivability and reduced immunogenicity, and there is a concern that the attenuated strain may be converted to wild-type Salmonella due to mutation and cause sepsis, which is a very important problem to be solved for actual use in cancer treatment. is recognized as

Strains such as Shigella , Vibrio cholera , E. coli , and Bifidobacterium , like Salmonella, penetrate cells of the intestinal tract and are targeted to cancer cells. Since these strains have low pathogenicity, they have the advantage that they can be used safely without concern for sepsis. Accordingly, studies are being conducted to develop strains capable of diagnosing cancer or expressing or suppressing substances useful for anticancer treatment, and using them for anticancer treatment.

For example, Patent Registration No. 10-2110993 discloses a composition for preventing, preventing or treating cancer containing a Klebsiella aerogenes strain as an active ingredient. However, unlike Salmonella, these strains do not reach the liver and spleen, which are important organs that cause an immune response, and thus have a relatively low effect on inducing systemic immunity.

E. coli Nissle (EcN) was discovered by Dr. Alfred Nissle (1874-1965) in 1917, and Shiga toxins (heat-stable ant heat-lavile toxines) were discovered through various safety tests over 100 years. , cytotoxins, invasiveness, pathogenic adhesion factors, hemolysis, serum resistance, and antibiotic resistance genes have been found to be absent. The non-pathogenicity of this strain is detoxified down to the genetic level, so it is safely used for various purposes.

ClyA (Cytolysin A) protein is a 34 kDa cytolytic protein expressed by E. coli , Salmonella and some intestinal bacteria, and kills target-cells by forming pores in the membrane. Attempts have been made to treat cancer by targeting ClyA to cancer cells using these apoptotic properties and then expressing it.

GM-CSF (Granulocyte-macrophage colony-stimulating factor) is a cytokine secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells and fibroblasts. GM-CSF induces the innate immune response by inducing the differentiation of monocytes into M1 type macrophages, while maturing into dendritic cells to interact with CD8 T cells to induce the acquired immune response, which is pivotal for the immune response. play a role GM-CSF expressed in bacteria has been used in various fields because its bioequivalence has been demonstrated in its function, but a series of processes are required to purify GM-CSF in the bacterial cytoplasm after expression.

Registered Patent No. 10-2110993

J. Mol. Med. 2010, 88, 763-773

An object of the present invention is to provide an anticancer strain capable of secreting proteins expressed by two different genes at the same time since it is equipped with a dual secretion system that operates with different mechanisms and does not affect each secretion.

Another object of the present invention is to provide an anti-cancer composition using the anti-cancer strain.

The present invention for achieving the above object is (A) a gene encoding a ClyA protein operably linked to a first inducible promoter; and (B) a gene construct operably linked to a second inducible promoter. It relates to an anticancer strain characterized in that it is transformed by a genetic construct comprising a; anti-sigma factor flgM gene and a gene encoding a GM-CSF protein linked thereto, and is equipped with a dual secretion system.

The present invention also relates to an anti-cancer composition containing the anti-cancer strain as an active ingredient.

As described above, according to the anticancer strain of the present invention, it is equipped with a dual secretion system and can simultaneously secrete the ClyA protein, which is a cell lytic protein, and the CM-CSF protein, which activates immunity, after targeting them to cancer cells. effect can be maximized.

In addition, when the anticancer strain of the present invention is a strain with low pathogenicity such as Escherichia coli or Bifidobacterium, the conventional strain can be safely and effectively used for anticancer treatment by supplementing the disadvantage of low immunity inducing effect.

1 is a schematic diagram of an anti-cancer strain according to an embodiment of the present invention.
2a is a conceptual map of the flgM-gmCSF-flhDC-pBad33 plasmid used in the examples of the present invention, and FIGS. 2b and 2c are the nucleotide sequence of the ClyA-flag constructed in the examples of the present invention and the constructed ClyA ( Conceptual map of the C-flag)-pBad18-asd+ plasmid.
Figure 3 is a photograph of Western blot results showing protein expression in double transformants according to an embodiment of the present invention.
Figure 4 is a photomicrograph showing macrophage morphological changes in the supernatant of the double transformant culture medium according to an embodiment of the present invention.
Figure 5 is a photograph of Western blot results showing the protein expression of macrophages by the supernatant of the double transformant culture medium according to an embodiment of the present invention.
Figure 6 is a photomicrograph showing the effect of treatment of the supernatant of the double transformant culture medium according to an embodiment of the present invention on cancer cells.
Figure 7 is a graph showing the killing effect of cancer cells by the supernatant of the double transformant culture medium according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and examples. However, these drawings and embodiments are only 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 obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical spirit of the present invention based on these examples. In addition, in describing the invention, if it is determined that a detailed description of a known technology related to the invention may unnecessarily obscure the subject matter of the invention, the detailed description will be omitted.

As described above, the present invention provides (A) a gene encoding a ClyA protein operably linked to a first inducible promoter; and (B) an anti-sigma factor operably linked to a second inducible promoter. A gene encoding a GM-CSF protein linked to the flgM gene; It relates to an anti-cancer strain characterized in that it is transformed by a genetic construct comprising a dual secretion system.

The strain is targeted to cancer during infection, and examples include Escherichia coli, attenuated Salmonella, Shigella, Acinetobacter, Pseudomonas aeruginosa, Listeria, Clostridium or intestinal microorganisms (beneficial bacteria), probiotics, but are limited thereto It is not. In the following examples, E. coli Nissle (EcN) was selected as a strain.

In the present invention, "attenuation" refers to artificially weakening the toxicity of living pathogens, mutating genes involved in essential metabolism of pathogens to induce immunity by stimulating only the immune system without causing disease in the body. For example, genes leading to attenuation of Salmonella are well known in the art, aroA, aroC, aroD, aroE, Rpur, htrA, ompR, ompF, ompC, galE, cya, crp, cyp, phoP, phoQ, rfaY, dksA, hupA, sipC, clpB, clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, relA and spoT It can be achieved by loss of function of one or more genes selected from the group consisting of: When applied in vivo, the functions of two or more of the above genes may be lost in order to prevent reduction to the wild type and further weaken toxicity.

As used herein, "inducible promoter" means a promoter capable of switching the operation of a gene linked by an inducer. The first inducible promoter and the second inducible promoter are each independently a tac promoter, a lac promoter, a lacUV5 promoter, an lpp promoter, a pLλ promoter, a pRλ promoter, a rac5 promoter, an amp promoter, a recA promoter, an SP6 promoter, a 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 pflE promoter, respectively. The first inducible promoter and the second inducible promoter can be operated by the same inducer, and the dual secretion system can be operated simultaneously by treating only one inducer, so it can be used more conveniently. there is. Alternatively, the operation of the first inducible promoter and the second inducible promoter may be switched by different inducers. In this case, there is an advantage in that the dual secretion system can be sequentially switched one by one, so that it can be used more safely.

One or more tags may be bound to the gene encoding the ClyA protein. It was confirmed in the following examples that the tag does not affect the anticancer activity of the ClyA protein. In order to facilitate detection when a tag is bound to the ClyA protein, for example, whether or not the ClyA protein is normally secreted after being targeted to a cancer site by labeling with a fluorescent material, and how long the secretion of the ClyA protein lasts in the body, it is easy. can be verified. In addition, by using the characteristics of the strain targeting cancer, it can be used not only for cancer treatment but also for tracking the treatment process by simultaneously checking whether the size of the cancer is reduced. Tags that may be used include, for example, His tag, Flag tag, Myc tag, HA tag, and GST tag, but are not limited thereto.

The tag linked to the gene encoding the ClyA protein has additional utility when the operation of the first inducible promoter and the second inducible promoter is switched by different inducers. That is, after targeting the anticancer strain to a cancer site, the first inducible promoter may be switched to operate by first treating the first inducing substance to secrete the ClyA protein having an anticancer effect. The secreted ClyA not only exhibits an anticancer effect through cytolytic activity, but also can be confirmed that it is targeted to the cancer site by detecting it. Thereafter, the second inducible promoter can be switched to operate by treating with a second inducing substance, thereby secreting the GM-CSF protein and exhibiting an additional immuno-anticancer effect. The anticancer strain of the present invention can be used in various ways depending on conditions such as this order or reverse order or simultaneous treatment.

The gene encoding the GM-CSF protein linked to the anti-sigma factor flgM gene is characterized in that it encodes a fusion protein of flgM and GM-CSF (hereinafter referred to as "flgM-GMCSF"), and a gene construct containing the gene The strain transformed by secretes flgM-GMCSF, which has the immune-inducing ability of GM-CSF, out of the cell. Therefore, the strain does not die due to the expressed GM-CSF and can stably and continuously express/secrete the fusion protein.

The linkage between the anti-sigma factor flgM gene and the gene encoding the GM-CSF protein may be directly coupled to the gene encoding the GM-CSF protein or linked through a linker. For example, the gene of SEQ ID NO: 1 linked via a linker having a gene sequence encoding 14 amino acids may be used, but is not limited thereto, and it will be easy for those skilled in the art to appropriately modify the length or sequence of the linker. .

In order to improve expression efficiency, a shine-dalgarno sequence may be arranged at the front end of the gene construct of the present invention. In addition, it is natural that appropriate restriction enzymes can be placed at the front and rear ends so that the gene construct of the present invention can be introduced into the vector. It is preferable that the flhDC gene is included in the genetic construct transforming the recombinant strain of the present invention. The flhDC gene is known to regulate the expression of the flgM gene as a major regulator of the flagellin operon gene. Accordingly, as a result of including the flhDC gene in the gene construct, it was confirmed that the expression level of flgM-GMCSF was greatly increased.

In the present invention, the transformation method follows a conventional method, and for example, a calcium chloride method, an electroporation method, etc. may be used, but is not limited thereto.

In the anticancer strain of the present invention, since the secretion mechanisms of the ClyA protein and the flgM-GMCSF protein expressed by each gene construct are different, that is, the dual secretion system is installed, the secretion of each is not affected. In addition, the secreted ClyA protein directly kills cancer cells, and the flgM-GMCSF protein exerts anticancer efficacy in a dual mechanism by inducing an immune response by influencing immune cells in the tumor microenvironment.

In the following examples, ClyA protein is well secreted out of Escherichia coli Nissle 1917, which is an enteric microorganism, without any other additional treatment, that there is no change in protein activity even when FLAG is tagged to the ClyA C-termianl, and that the secreted protein dissolves and kills cancer cells. It was confirmed that it induces immune cells better.

In addition, in the following examples, the flgM-GMCSF fusion protein was induced by Arabinose and secreted well out of Escherichia coli Nissle 1917, the function of the secreted protein was the same as that of GM-CSF, and the secreted protein induces immune cells to produce cancer cells It was confirmed that the pBad33 plasmid is well maintained in cells as a low copy plasmid.

According to the present invention, since the origin of the plasmid introduced into the transformant is different, the two plasmids can exist independently, and the flgM-GMCSF and ClyA contained in each plasmid have different secretion mechanisms outside the transformant strain. Since they do not affect each other's secretion, it is possible to maximize efficacy, such as affecting immune cells in the tumor microenvironment by flgM-GMCSF and directly killing cancer cells by ClyA.

In addition, by inducing the expression of two plasmids as one derivative or individual derivatives as needed, various response strategies can be selected according to the use environment.

In addition, safety can be secured by adopting Escherichia coli E. coli Nissle 1917, an intestinal microorganism whose toxicity is guaranteed, as a host cell, as in the following examples.

The present invention also relates to an anti-cancer composition containing the anti-cancer strain as an active ingredient. After administration of the strain, when the strain is targeted to cancer tissue, a drug that induces the operation of the inducible promoter is administered to simultaneously or sequentially induce the secretion of the ClyA protein and the flgM-GMCSF fusion protein from the strain. .

The composition of the present invention can be prepared by a method known in the pharmaceutical field to be used as an anti-cancer 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 into a preparation for oral or parenteral administration. The dosage of the active ingredient according to the present invention may be appropriately selected depending on the degree of absorption of the active ingredient in the body, the form of the preparation, the age, sex and condition of the patient, the degree of symptoms, etc., administration may be administered once a day, , It may be administered in several divided doses. A typical dosage is 0.001 mg/kg·day to 10 g/kg·day.

[Example]

1. Plasmid preparation and transformant construction

(1) Securing the plasmid cloned with the GM-CSF gene

In flgM-gmCSF-flhDC-pBad33 / ppGpp- (accession number: KCTC14455BP) used in 'gene construct for secretion of GM-CSF and anti-cancer recombinant strain transformed thereby' filed under Application No. 10-2021-0010768 The flgM-gmCSF-flhDC-pBad33 plasmid (cm) was extracted in a conventional manner to secure the plasmid used for GM-CSF secretion. A conceptual map of the flgM-gmCSF-flhDC-pBad33 plasmid is shown in Figure 2a.

(2) Cloning a plasmid having the ClyA gene

Based on the nucleotide sequence (NCBI) of the clyA gene of Escherichia coli MG1655 K-12 (wild species), NheI and HindIII sites were inserted before and after the clyA gene for cloning, respectively, and a flag tag (red) was inserted at the C-terminal to obtain ClyA -flag was synthesized (SEQ ID NO: 2). This process was done through the gene synthesis service of a professional company (Cosmogenetech Co., Ltd.). Base sequence information of the prepared ClyA-flag is shown in FIG. 2B.

The synthesized ClyA-flag plasmid was treated with 10 U each of NheI (Takara Co., Ltd.) and HindIII (Takara Co., Ltd.) at 37°C for 2 hours, and electrophoresis was performed. About 1000 bp of DNA was extracted from the electrophoresis gel (Gel extraction kit: Qiagen) to secure the ClyA insert.

pBad18 asd+ plasmid (distributed from the laboratory of Professor Heon-Man Lim, Chungnam National University) was reacted at 37℃ for 2 hours using 10U each of NheI (Takara) and HindIII (Takara), and then the reaction product was purified with a PCR purification kit (Qiagen) to obtain VECTOR DNA was prepared.

Prepared ClyA Insert and VECTOR DNA were ligated at 25℃ for 30 minutes (Invitrogen Co., Ltd.), and then transformed into DH5a competent cells (distributed from the laboratory of Prof. Heon-Man Lim, Chungnam National University), smeared on LB amp (containing ampicillin) solid medium, and cultured to form colonies was cultured.

Six of the cultured colonies are selected, cultured in LB amp (containing ampicillin) liquid medium at 37° C. for 16 hours, and then DNA is extracted. Afterwards, NheI (Takara Co., Ltd.) and HindIII (Takara Co., Ltd.) were reacted for 1 hour at 10 U and 37 ℃, followed by electrophoresis to confirm the 1000 bp band, and the base sequence was analyzed (Cosmogenetech Co., Ltd.) to ClyA (C-flag) -pBad18 -asd+ plasmid was constructed. A conceptual map of the constructed ClyA(C-flag)-pBad18-asd+ plasmid is shown in Figure 2c.

For reference, the asd gene encodes the enzyme aspartate-semialdehyde dehydrogenase. Diaminopimesan (DAP) is an essential component of peptidoglycan required for cell wall synthesis of gram-negative bacteria. Without DAP, asd-bacteria die rapidly. Since DAP does not exist in mammalian tissues, asd- mutant bacteria must carry the asd+ plasmid to survive. pBAD18 asd- plasmid disappears from cells after about a day. To prevent this, pBad18 asd+ plasmid was used for E. coli Nissle 1917 asd- strain.

(3) Production of transformants

The flgM-gmCSF-flhDC-pBad33 plasmid and the ClyA(C-flag)-pBad18-asd+ plasmid obtained above were simultaneously transformed into E. coli Nissle 1917 asd- (distributed from Professor Lim Heon-man, Chungnam National University) and LB amp, cm (ampicillin, chloramphenicol) and plated on solid medium to obtain ClyA(c-flag)-pBad18-asd + flhDC+flgM-(his)-GMCSF-pBad33 / E. coli Nissle 1917 asd- (hereinafter abbreviated as 'double transformant'). selected. The prepared double transformant was deposited and assigned accession number KCTC19041P.

2. Characterization of double transformants

(1) Check protein secretion

After inoculating the double transformant single colony into LB amp, cm liquid medium, incubating at 37℃ (the culture temperature below is the same) in a shaking incubator for 12-16 hours, the cells are incubated in a new LB amp, cm liquid medium with an OD600 of 0.05. After matching, it was cultured with shaking at 37 ° C. for 2 hours.

When the OD600 was 0.4-0.6, the arabinose was divided into untreated control (-) and treated treatment (+) at a concentration of 0.2% (final), and cultured with shaking for 5 hours, and samples were collected.

After centrifuging 1ml (1, 2 lanes) of whole cell culture medium and another 1ml culture medium, samples were prepared by separating pellets (3, 4 lanes) and supernatant supernatant (5, 6 lanes). The supernatant was again completely free of bacteria by 0.2um filtering and concentrated using pall's nanosep (OD010C35 3K omega).

50ul of each obtained sample was mixed with SDS sample buffer to make a sample for Western blotting. The sample was denatured on a 100°C heat block for 5 min, centrifuged at 13000 rpm, 5 min, and 4°C, and electrophoresed on a 10% polyacrylamide gel. The gel was transferred using polyvinylidene fluoride (PVDF) to transfer proteins to a PVDF membrane. The membrane was blocked for 1 hour at room temperature using a blocking buffer. Afterwards, the primary antibody anti-his tag (SB194b, SouthernBiotech) was used to identify flgM-gmCSF, the anti-flag tag (SAB4200071, Millipore) to identify ClyA, and the anti-dnaK (ab69617) to identify DnaK. , abcam) were each diluted 1/1000 and reacted at 4°C for 16 hours. Then, the membrane was washed with TBST (Tris-buffered saline with 0.1% Tween-20) three times at 10-minute intervals, and mouse anti-mouse-IgG (#7076s, Cell signaling, Danvers, MA, USA) secondary antibody and rat anti-rat-IgG (#7077, Cell signaling, Danvers, MA, USA) were reacted at room temperature for 1 hour. After the reaction, it was washed three times with TBST at 10-minute intervals, exposed to X-ray films using ECL buffer, and then developed to confirm the expression level of each protein (see FIG. 3).

As can be seen in Figure 3, in the double transformant according to the present invention, ClyA and flgM-GMCSF are induced by Arabinose (whole cell (1,2 lanes) and pellets (3,4 lanes)), and they are well outside the strain. secreted (5, 6 lanes).

However, in the case of Western blotting with anti-dnaK, DnaK was detected in whole cells and pellets regardless of the presence or absence of induction. On the other hand, Dnak was not detected in the supernatant group. DnaK is a molecular chaperone belonging to the Hsp70 family and is evenly distributed throughout bacteria. The fact that DnaK was found in whole cells and pellets but not in the supernatant indicates that the corresponding proteins ClyA and flgM-GMCSF are made. This shows that the cells stably secrete ClyA and flgM-GMCSF, respectively, while surviving without bursting.

(2) Activity test of secreted flgM-GMCSF

① Treatment of supernatant to macrophages

RAW264.7 cells, a mouse macrophage cell line, were purchased from Korea Cell Line Bank (KCLB), and DMEM medium containing 10% FBS and 1% streptomycin-penicillin was used for cell culture. Cells were cultured at 37°C and 5% CO2 conditions.

Separately, the double transformant single colony was inoculated into LB amp, cm liquid medium, cultured in a shaking incubator at 37 ° C for 12-16 hours, and cells were adjusted to OD600 of 0.05 in new LB amp, cm liquid medium. 37 It was cultured with shaking for 2 hours at ℃.

When the OD600 is 0.4-0.6, the arabinose is divided into untreated control (-) and treated (+) at a concentration of 0.2% (final), cultured with shaking for 5 hours, and then the culture medium is centrifuged at 3000 rpm to obtain the supernatant. separated. The bacteria were completely removed from the supernatant by 0.2um filtering to prepare ' (-) supernatant ' and ' (+) supernatant '.

When the mouse macrophage cell line, RAW264.7 cells, was 2×10 ⁵ , the supernatant obtained above was treated with 200ul each and cultured for 24 hours.

② Morphological changes of macrophages by supernatant treatment

After culturing, the results were observed under a microscope at 200x magnification (see FIG. 4). As can be seen in Figure 4, RAW264.7 cell had a round shape before differentiation (1), and when treated with (-) supernatant, which was not expressed by arabinose (2), some cells were transformed into a long shape was formed, and when the supernatant (+) expressed by arabinose was treated (3), more than 50% of the cells were differentiated into elongated or star-shaped cells. That is, it can be seen that RAW264.7 cells are morphologically differentiated into M1 type macrophages by the (+) supernatant according to the present invention. From this, it was confirmed that the flgM-GMCSF prepared from the double transformant according to the present invention has the same immunity-inducing activity as GMCSF.

③ Immunological changes in macrophages by supernatant treatment

RAW264.7 cells treated with (+) and (-) supernatants and cultured for 24 hours were washed twice with PBS, lysed with RIPA buffer, and centrifuged at 13000 rpm, 20 min, 4 ° C to obtain supernatants. Got it. The protein in the quantified supernatant was mixed with SDS sample buffer at a concentration of 1 ug/1 ul, denatured in a 100 ° C heat block for 5 min, and then centrifuged at 13000 rpm, 5 min, 4 ° C. The centrifuged material was electrophoresed on a 10% polyacrylamide gel, and the gel was transferred using PVDF (polyvinylidene fluoride) to transfer proteins to a PVDF membrane. Subsequently, the membrane was blocked for 1 hour at room temperature using a blocking buffer, and then anti-mouse CD80 (#sc-376012; Santa Cruz, Dallas, TX, USA) and anti-mouse CD86 (#sc-28347; Santa Cruz, Dallas, TX, USA) and anti-rabbit GAPDH (#sc-25778; Santa Cruz, Dallas, TX, USA) were added and reacted at 4°C for 16 hours. Then, the membrane was washed with TBST (Tris-buffered saline with 0.1% Tween-20) three times at 10-minute intervals, and then anti-rabbit (#7074s, Cell signaling, Danvers, MA, USA) or anti-HRP-coupled membrane was used. -mouse-IgG (#7076s, Cell signaling, Danvers, MA, USA) secondary antibody was reacted at room temperature for 1 hour. Subsequently, the membrane was washed three times with TBST at 10-minute intervals, and then exposed to an X-ray film using ECL buffer and developed to confirm the expression level of each protein.

Referring to Figure 5, the expression levels of CD80 and CD86, known as M1 Macrophage markers, were significantly increased in the arabinose-induced (+) supernatant treatment group, and it was found that RAW264.7 cells were more polarized to M1 type by flgM-GMCSF. can

Therefore, the flgM-GMCSF secreted by the double transformant ClyA(c-flag)-pBad18-asd + flhDC+flgM-(his)-GMCSF-pBad33 / E. coli Nissle 1917 asd- according to the present invention is similar to normal GMCSF. It was confirmed that they exhibited the same activity. This, together with the results in ② above, proves that the double transformant according to the present invention is an excellent strain for inducing immunity.

(3) Activity test of secreted ClyA

① Treatment of supernatant to cancer cell line

After inoculating the double transformant single colony into LB amp, cm liquid medium, incubating at 37℃ (the culture temperature below is the same) in a shaking incubator for 12-16 hours, the cells are incubated in a new LB amp, cm liquid medium with an OD600 of 0.05. After matching, it was cultured with shaking at 37 ° C. for 2 hours.

When the OD600 is 0.4-0.6, the arabinose is divided into untreated control (-) and treated (+) at a concentration of 0.2% (final), cultured with shaking for 5 hours, and then the culture medium is centrifuged at 3000 rpm to obtain the supernatant. separated. The supernatant was completely removed by 0.2um filtering again.

Two types of human colorectal cancer cell lines, HT-29 and SW480 (both American Type When the number of cells was 2 × 10 ⁵ , the supernatant obtained above was concentrated 50 times, treated with 200 μl each, and cultured for 24 hours.

② Microscopic observation of cancer cells by supernatant treatment

After culturing, the results were observed under a microscope at 100x magnification (see FIG. 6). As can be seen in FIG. 6, in the (-) supernatant treatment group not treated with Arabinose, about 5% of the cells were empty compared to the control group (No treat) in which nothing was treated, but in the (+) supernatant treatment group treated with Arabinose, the cells were empty. Since about 30% of the (+) supernatant is empty, it can be inferred that the supernatant inhibits the growth of cancer cells or kills cancer cells.

As a result, the ClyA protein secreted by the double transformant of the present invention, ClyA(c-flag)-pBad18-asd + flhDC+flgM-(his)-GMCSF-pBad33 / E. coli Nissle 1917 asd-, is effective against human colorectal cancer 2 It was confirmed that the cell lines HT-29 and SW480 were inhibited or killed.

③ Confirmation of death of cancer cells by supernatant treatment

Cell death is divided into apoptosis, which is a mechanism in which proliferation is stopped, cells shrink, and DNA is broken down into regular fragments, and necroptosis, in which cells swell and membranes burst, releasing internal components to the outside of the cell. ClyA is known to act as a necroptosis that kills cells by forming PORE in the cell membrane.

In order to confirm cell death of HT29, SW480, and CT26 cultured in (3) ① above, CCK assay and LDH assay (experiment to confirm necroptosis) were performed as follows, and the results are shown graphically in FIG. 7 .

First, culture supernatants (cancer cell culture supernatants) of No treat, (-) supernatant treatment group ('Arabinose-' in the figure), and (+) supernatant treatment group ('Arabinose+' in the figure) were taken, respectively, and CCK assay: CCK: Apoptosis was confirmed using DogenBio (ez-500, EZ-Cytox) and EZ-LDH Cell cytotoxicity assay kit (DG-LDH500, DoGEN).

For the CCK assay, 100ul of the culture supernatant was transferred to a 96-well plate and 100ul of CCK solution was added.

For the LDH assay, 10ul of the culture supernatant was put in a 96-well plate, 100ul of EZ-LDH Cell cytotoxicity solution was added, and the mixture was reacted at room temperature for 30 minutes.

Each was measured at a wavelength of 450 nm using a Microplate reader. Experiments were conducted in triplicate.

As can be seen in Figure 7, it was confirmed through the CCK assay that the apoptosis effect of HT29, SW480 and CT26 was superior to no treat or arabinose- in the arabinose+ treatment group. In addition, in the LDH assay, the arabinose+ treatment group confirmed a value of about 150% or more than the No treat control group, and it was also confirmed that ClyA induces apoptosis by lysing and killing cells (Necroptosis) as known.

These results show that ClyA has the effect of directly dissolving and killing cancer cells, and the internal components of cancer cells that are dissolved and released to the outside stimulate immune cells to induce secondary anti-cancer immunity effects.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC19041P 20221020

Claims (10)

(A) a genetic construct comprising a gene encoding a ClyA protein operably linked to a first inducible promoter;
(B) a gene encoding a GM-CSF protein linked to an anti-sigma factor flgM gene operably linked to a second inducible promoter; transformed by a genetic construct comprising,
An anticancer strain characterized by being equipped with a dual secretion system.
The method of claim 1,
The anticancer strain, characterized in that the strain is selected from attenuated Salmonella, Escherichia coli, Shigella, Acinetobacter, Pseudomonas aeruginosa, Listeria, Clostridium or intestinal microorganisms, probiotics.
The method of claim 2,
The strain is an anti-cancer strain, characterized in that E. coli Nissle 1917.
The method of claim 2,
The first inducible promoter and the second inducible promoter are each independently a tac promoter, a lac promoter, a lacUV5 promoter, an lpp promoter, a pLλ promoter, a pRλ promoter, a rac5 promoter, an amp promoter, a recA promoter, an SP6 promoter, a 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 An anticancer strain characterized in that it is selected from the group consisting of a promoter, a pagC promoter, a hip promoter, ansB promoter and a pflE promoter.
The method according to any one of claims 1 to 4,
The recombinant strain, characterized in that the gene encoding the ClyA protein is bound to one or more tags.
The method according to any one of claims 1 to 4,
The linkage between the anti-sigma factor flgM gene and the gene encoding the GM-CSF protein is characterized in that the flgM gene and the gene encoding the GM-CSF protein are directly coupled or connected through a linker.
The method of claim 6,
A recombinant strain characterized in that the gene encoding the GM-CSF protein linked to the anti-sigma factor flgM gene consists of SEQ ID NO: 1.
The method of claim 7,
The strain is a recombinant strain, characterized in that ClyA (c-flag) -pBad18-asd + flhDC + flgM- (his) -GMCSF-pBad33 / E. coli Nissle 1917 asd- (KCTC19041P).
The method according to any one of claims 1 to 4,
The genetic construct is a recombinant strain, characterized in that it comprises a flhDC gene.
An anti-cancer composition comprising the anti-cancer strain according to any one of claims 1 to 4 as an active ingredient.

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