KR102224855B1 - Anticancer Salmonella Bio-synthesizing and Secreting Indirubin 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 inducing immune stimulation along with biosynthesis and secretion of indirubin having anticancer activity by being targeted to a tumor site, and an anticancer composition comprising the same. More particularly, the present invention relates to an attenuated anti-cancer Salmonella strain that is transformed with a gene construct including a flavin-containing monooxygenase (fmo) gene and a tryptophanase (tnaA) gene to produce indirubin at a cancer site, and an anti-cancer composition comprising the same.

Description

TECHNICAL FIELD [Anticancer Salmonella Bio-synthesizing and Secreting Indirubin and Anticancer Composition Comprising the Salmonella]

The present invention relates to a novel Salmonella strain that can be effectively used for anticancer therapy by biosynthesis and secretion of indirubin, which is targeted to the tumor site and has anticancer activity, and induces immune stimulation, and an anticancer composition comprising the same.

Cancer is a life-threatening disease in the end by destroying normal cells by penetrating into surrounding tissues as cells do not stop proliferating. As the human body's ability to regulate due to aging decreases, it becomes susceptible to cancer, and in an aging society, cancer occupies the unchanged number one cause of death for 10 years since 2007, exceeding twice the mortality rate of heart disease, the second highest. .

The most effective response system for the treatment of diseases is to strengthen immunity, but cancer cells are not external invaders, so they do not induce the body's immune response and avoid. Certain viruses or bacteria are more likely to infect cancer cells than normal cells, and infected bacteria can be targeted by immune cells. Accordingly, anticancer treatment methods have been proposed that deliberately infect specific viruses or bacteria to stimulate the body's immune response to fight cancer cells. Infectious bacteria such as Shigella, Vibrio cholera, and pathogenic E. coli only penetrate the cells of the intestine, but do not reach the liver and spleen, which are important organs that trigger an immune response. In contrast, Salmonella can infiltrate the spleen and liver through lymph nodes and stimulate systemic immune responses. Specifically, Leschner et al. (J. Mol. Med. 2010, 88, 763-773) infected mice with CT26 tumors with a fluorescent Salmonella intravenously and followed the infection route over time. . As a result, it was shown that the whole body was infected through the blood of the mouse immediately after infection, and Salmonella was accumulated in the spleen and liver after 20 minutes of infection, and it was observed that Salmonella was intensively accumulated only in the tumor tissue after 24 hours.

However, Salmonella is a representative bacteria that cause food poisoning, and it is too pathogenic to be used directly for cancer treatment because it can cause sepsis by infection and threaten life. A research team at Yale University in the United States announced that if Salmonella is genetically engineered, it can be attenuated by removing only toxicity while maintaining the characteristics of attacking tumors, and it can suppress tumors by inducing immune stimulation through injection of the attenuated Salmonella.

Thereafter, studies were conducted to develop strains capable of additionally expressing or inhibiting substances useful for chemotherapy along with attenuation and immune activation, and to use them for chemotherapy. Registered Patent No. 10-0852687 has been published on a Salmonella strain expressing tumor necrosis factor alpha transduced with a tumor necrosis factor alpha protein vector in an attenuated Salmonella strain, and a composition for anticancer treatment containing the same. -1544602 was published on the Salmonella strain expressing inhibin alpha shRNA and an anticancer agent composition containing the same.

On the other hand, indirubin as an active ingredient of Angelica Angela drew attention as it was reported that it was effective against chronic granular leukemia in the 1980s. Similar to standard treatment with busulfan, an inhibitor of cellular activity, more than 50% of patients treated with indirubin were partially or completely remission, with low toxicity and mild side effects. Accordingly, many studies are being conducted to use indirubin and its derivatives as anticancer agents.

Indirubin is an isomer of indigo and is only produced in a small amount as a by-product in the industrial production process of indigo, but industrially, mass production is not achieved. In addition, since chemical synthesis is harmful to the environment, biosynthesis using microorganisms is mainly performed. Registered Patent No. 10-0173046 reported that indirubin can be produced by adding indole during cultivation of P. tinctorium strain, and Patent No. 10-1092515 discloses recombinant E. coli containing a monooxygenase gene. It has been reported that tryptophan can be converted to indirubin. In addition, Patent No. 10-1021789 reported that indirubin can be produced from indican using a specific strain of wild-type E. coli. However, in the prior art, it was reported that Salmonella had very low indirubin production efficacy under the same conditions at the same time.

Registered Patent No. 10-0852687 Registered Patent No. 10-1544602 Registered Patent No. 10-0173046 Registered Patent No. 10-1092515 Registered Patent No. 10-1021789

Leschner et al. (J. Mol. Med. 2010, 88, 763-773)

The present invention is used for anticancer treatment by transforming Salmonella, which has characteristics targeted to cancer, but does not produce indirubin, which has anticancer properties, is targeted to the cancer site, stably expresses indirubin, and has immune-inducing activity. It is an object of the present invention to provide a new Salmonella strain that can be used.

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 that is transformed with a genetic construct including a flavorin-containing monooxygenase (fmo) gene and a tryptophanase (tnaA) gene to produce indirubin at a cancer site.

Since genes artificially inserted by transformation are not essential for the survival of bacteria, plasmids are removed during in vivo proliferation, and the attenuated Salmonella strains may lose the ability to produce indirubin. Accordingly, in order to stably maintain the production of indirubin by the plasmid, the attenuated Salmonella strain has a deletion of the glmS gene, and the plasmid preferably contains the glmS gene. Due to this structure, since the glmS gene expressed by the plasmid is essential for the survival of Salmonella strains, the plasmid can be stably maintained in vivo.

It has been reported that E. coli can be used for the production of indigo or indirubin after the introduction of the wild type itself or the monooxygenase gene, but it is known that the production of Salmonella, which is evolutionarily close to E. coli, is extremely small. In the following examples, when only the monooxygenase (fmo) gene was introduced, the production of indigo was confirmed in E. coli, but the production of indigo or indirubin was not confirmed in Salmonella. However, when the tnaA gene was introduced together with the fmo gene, it was confirmed that the Salmonella strain produced indirubin.

According to the present invention, when Salmonella is infected in the body, it is targeted to the tumor site about 24 hours after and induces immunity to exhibit an anticancer effect, and indirubin is produced and secreted at the tumor site in the body to exert an additional anticancer effect by indirubin. . In the present invention, in order to be able to apply the Salmonella strain to the body, it is necessary to ensure safety due to infection of the Salmonella strain. To this end, the present invention is characterized in that the attenuated Salmonella strain is transformed with a plasmid containing the fmo gene and the tnaA gene.

In the present specification, "attenuated" refers to artificially weakening the toxicity of a living pathogen, and does not cause disease in the body by mutating genes involved in essential metabolism of pathogens, and means inducing immunity by stimulating only the immune system. Salmonella bacteria have serious effects such as typhoid disease or food poisoning when infected with humans, so they must be attenuated in order to be applicable to the human body. Salmonella attenuation is well known in the art, aroA, aroC, aroD, aroE, Rpur, htrA, ompR, ompF, ompC, galE, cya, crp, cyp, phoP, phoQ, rfaY, dksA, hupA, sipC, Selected from the group consisting of clpB, clpP, clpX, pab, nadA, pncB, pmi, rpsL, hemA, rfc, poxA, galU, cdt, pur, ssa, guaA, guaB, fliD, flgK, flgL, flhDC, relA and spoT It can be achieved by the loss of function of one or more genes.

Although the fmo gene and the tnaA gene were simultaneously introduced into the attenuated Salmonella strain, the indirubin production efficiency was different depending on the type of the attenuated Salmonella strain. Whether the glmS gene was deleted in the attenuated strain also affected the indirubin production efficiency. When the attenuated strain was a strain in which the functions of galE and flhDC genes were lost, the production efficiency of indirubin was particularly excellent. In particular, when the attenuated Salmonella strain in which the glmS gene was deleted together with the galE and flhDC genes was transformed with a plasmid containing the fmo+tnaA+glmS gene, the same plasmid was used to transform E. coli in which the glmS gene was deleted. It was shown that the ability to produce indirubin was excellent even compared to the strain.

Accordingly, the production strain of the example in which the following indirubin production was quick and efficient was named fmo-tnaA-glmS/galE flhDC glmS Salmonella, and was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on March 20, 2020 (accession number KCTC18809P). .

The present invention also relates to an anticancer composition containing the Salmonella strain as an active ingredient.

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, and mixed with itself or a pharmaceutically acceptable carrier, an excipient, a diluent, etc. Can be used.

The composition of the present invention can 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 depending on the degree of absorption of the active ingredient in the body, the form of the formulation, the age, sex and condition of the patient, the degree of symptoms, etc., and the administration may be administered once a day. Alternatively, it may be administered several times. Typical dosage is 0.001mg/kg·day~10g/kg·day.

As described above, according to the Salmonella strain according to the present invention, the attenuated Salmonella strain is attenuated to induce an immune response at the tumor site to exhibit an anticancer effect, and at the same time, indirubin is produced/secreted to achieve the anticancer effect by indirubin. As it can be represented, it can be more usefully used for anticancer treatment.

1 is a cleavage map of pBLUE1.7 used in an embodiment of the present invention.
Figure 2 is a schematic diagram of the pBLUE fmo+tnaA plasmid used in an embodiment of the present invention.
Figure 3 is a photograph of assaying the production of indirubin of strains transformed with fmo+tnaA plasmid.
Figure 4 is a photograph and analysis results showing the production of indirubin fmo + tnaA / KST0650 strain.
5 is a schematic diagram of the pUC fmo+tnaA+glmS plasmid used in an example of the present invention.
Figure 6 is a photograph of testing the production of indirubin of the strain transformed with pUC fmo+tnaA+glmS.
Figure 7 is a schematic diagram of fmo+tnaA+glmS/galE flhDC glmS- Salmonella strain.
Figure 8 is a photograph showing the production of indirubin fmo+tnaA+glmS/galE flhDC glmS- strain.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, these embodiments are only examples for easily explaining the content and scope of the technical idea of the present invention, thereby not limiting or changing the technical scope of the present invention. It will be obvious to those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention based on these examples.

[Example]

Example 1: Indirubin production assay of recombinant strains containing fmo gene

The fmo (flavin-containing monooxygenase) gene of M. amonisulfidivorans is a gene that converts indole to indigo or indirubin. The most efficient part is the upper DNA containing the fmo gene, which is 300 bp, which is 1.7 kb. Plasmid pBLUE1.7 (Registration Patent No. 10-1092515, Fig. 1) in which the 1.7 kb fmo gene was inserted into the pBLUEscript SK(+) vector was distributed and used from the laboratory of Professor Kim Si-wook of Chosun University.

After transforming various strains using the pre-sold plasmid, in order to test whether the transformed strain produced indigo or indirubin, it was plated on LB amp solid medium and cultured at 37°C for 24 hours. It was confirmed that E. coli strains D5a and MG1655 transformed with pBLUE1.7 formed blue colonies and mainly produced indigo, and DH5a produced indigo faster than the MG1655 strain. However, wild-type Salmonel lines LT2 and 14028S and attenuated Salmonel lines aroA-, ppGpp-, and KST0650 all showed no color change during cultivation after transformation, indicating that indigo or indirubin was not produced.

Example 2: Construction of a recombinant strain containing the fmo+tnaA gene and assay for indirubin production

In order to obtain the tnaA gene, PCR was performed using the chromosomal DNA of MG1655, a wild species E. coli, as a template, using the primer pair in Table 1 below and PrimeSTAR Max DNA Polymerase 2X master mix (Takara Co., Ltd.). PCR conditions were 98° C. 3 minutes and 35 cycles of 98° C. 10 seconds-55° C. 5 seconds-72° C. 1 minute 30 seconds and 75° C. 3 minutes. The PCR product was reacted with 10U of Hindlll (Takara Co., Ltd.) at 37°C for 1 hour, followed by electrophoresis, and DNA was extracted from the electrophoresis gel using a Gel extraction kit (Qiagen), and 1.5 kb of DNA was extracted. tnaA insert was prepared

After 1 μg of pBLUE 1.7 plasmid was reacted with 10U of Hindlll at 37°C for 2 hours, electrophoresis was carried out in the same manner as in the preparation of tnaA insert, and a pBLUE1.7 vector was prepared through gel extraction.

After the tnaA insert prepared on pBLUE and the pBLUE1.7 vector were ligated at 25° C. for 30 minutes (Invitron, Inc.), DH5a competent cells were transformed. Transformed DH5a cells were plated on LB amp solid medium and cultured at 37°C to select colonies having antibiotic resistance. Among the selected colonies, 6 candidates were reacted with 10 U of Hindlll (Takara Co., Ltd.) for 1 hour at 37°C, and then the generation of a 1.5 kb band was confirmed through electrophoresis. By requesting Cosmogenetech to analyze the nucleotide sequence of the candidate, the insertion of the tanA gene was confirmed, and the fmo+tnaA plasmid was established (FIG. 2).

After transforming various strains in the same manner as in Example 1 using fmo+tnaA plasmid, the transformed strain was tested for production of indigo or indirubin. It was confirmed that the transformed E. coli strains D5a and MG1655 formed blue colonies and mainly produced indigo. Wild-type Salmonel line LT2 and 14028S strains also formed blue colonies, indicating that indigo was mainly produced. 3 is a photograph showing the culture results of transformed attenuated Salmonella, 1 and 2 are the culture results of the transformed KST0650 strain, and 1 is the culture results in a medium to which tryptophan is added. 3 is a transformed araA- strain, and 4 is a transformed ppGpp glmS- strain. The KST0650 strain, the araA- strain, and the ppGpp glmS- strain were dispensed from the labs of Jeongeup Atomic Energy Research Institute, Professor Insoo Lee of Hannam University, and Professor Jaeho Jeong of Chonnam University, respectively. In Figure 3, it can be seen that the araA- and ppGpp glmS- strains produce indigo together with indirubin by transformation, and the KST0650 strain mainly produces indirubin, and indirubin production is further enhanced by the addition of tryptophan. Able to know.

In order to more clearly confirm the production of indigo and indirubin, fmo+tnaA/KST0650 strain, which was confirmed to have good indirubin production efficiency, was inoculated into 50 mL of a liquid medium, and then under conditions of 150 rpm or 250 rpm at 37°C. Incubated for 24 hours. After taking 500 µl of the culture medium, centrifugation was performed to remove the supernatant, and the cell pellet was dissolved in DMSO. Commercially available indirubin (Sigma) was used as a standard and the content and concentration of indirubin in the solution were calculated by HPLC. Figure 3 shows the dissolution picture and HPLC analysis results of the pellet, it can be seen that 14.53 mg/ml in 1 cultured at 150 rpm and 37.84 mg/ml indirubin in 2 cultured at 250 rpm.

From this, it was confirmed that the fmo+tnaA plasmid could be used to transform indirubin to produce indirubin from a Salmonella strain in which indirubin was not produced.

Example 3: Preparation and transformation of glmS- attenuated strain and assay for production of indirubin

In order to stably express the plasmid in vivo in the absence of antibiotics, it is recommended to insert the glmS gene into the plasmid and knock out the glmS gene in the strain. First, the glmS gene was knocked out from the attenuated strain.

Another glmS- attenuated strain was prepared by performing P22 transduction from the ppGpp glmS- strain. Specifically, 2 mL of P22 broth (0.2% glucose, 1% EX50 salts, P22 stock ~10 ¹¹ /mL) was added to 0.2 mL of ppGpp glmS- strain cultured with shaking at 37°C for 16 hours and incubated at 37°C for 9 hours. . The culture medium was centrifuged, the supernatant was transferred to a new tube, and treated with chloroform to prepare a ppGpp glmS- P22 lysate containing a glmS- mutation.

0.1 mL attenuated strain cultured with shaking at 37°C for 16 hours and the ppGpp glmS-P22 lysate were mixed in a volume ratio of 1:1 and reacted for 1 hour at room temperature (~25°C), and then 10 ⁰ , 10 ^{-1 in PBS solution} , 10 ^-2 and 10 ^-3 and plated on LB kanamycin plates. After selecting colonies that grew well in the kanamycine medium, the glmS- strain was established by requesting sequencing to Cosmogenetech.

As attenuated strains used for transduction, aroA- strain, KST050 strain, and galE flhDC- strain were used. To obtain galE fhlDC- strain, Salmonella typhimurium LT2 ΔgalE::tetRA, ΔflhDC::Tn10 (Patent Application No. 10-2020-0001602, KCTC18801P) was cultured in a liquid LB medium without ampicillin for 48 hours. The culture solution was centrifuged at 13000 rpm for 30 seconds, and the collected cells were plated on a solid medium and cultured. Each colony obtained after cultivation was inoculated in ampicillin solid medium (100 µg/ml) and tetracycline solid medium (12.5 µg/ml) and cultured to select colonies that do not grow in ampicillin medium but only in tetracycline medium to prepare galE flhDC- strains. .

The glmS- attenuated strains were transformed using the fmo+tnaA plasmid in the same manner as in Example 2, and then the production of indirubin was assayed. As a result, aroA- did not significantly affect the production of indirubin due to the knockout of glmS, whereas KST0650, which had the best indirubin production ability, hardly produced indirubin after the knockout of glmS. galE flhDC- has also been shown to produce indirubin.

Example 4: Construction of a recombinant strain containing the fmo+tnaA+glmS gene and assay for indirubin production

The glmS gene was inserted into the plasmid so that glmS was knocked out and the strain transformed with the fmo+tnaA plasmid could stably maintain the plasmid in vivo.

1) Construction of pUC fmo+tnaA plasmid

The fmo+tnaA plasmid was constructed using a pUC vector with a larger number of copies than the pBLUE vector. To this end, the fmo+tnaA plasmid prepared using the pBLUE vector in Example 2 was reacted with 10 U of HindIII and 10 U of BamHI (Takara, Inc.) for 1 hour at 37° C., and 3.2 kb of fmo was subjected to electrophoresis and gel extraction. +tnaA insert was produced.

The pUC plasmid was reacted with 10U of HindIII and 10U of BamHI for 1 hour at 37°C, and a 2.6 kb pUC vector DNA was prepared through electrophoresis and gel extraction, and the fmo+tnaA insert was ligation (Invitron) at 25°C for 30 minutes. Then, DH5a competent cells were transformed. The transformed cells were plated on LB amp solid medium and cultured at 37°C to select colonies having antibiotic resistance. Four candidates for the selected colonies were selected and reacted with HindIII 10U and BamHI 10U for 1 hour at 37°C, and then the generation of 3.2 kb band was confirmed through electrophoresis. By requesting Cosmogenetech to analyze the nucleotide sequence of the candidate, the insertion of the fmo+tanA gene was confirmed, and the pUC fmo+tnaA plasmid was established.

2) Construction of fmo+tnaA+glmS plasmid

The pUC fmo+tnaA plasmid prepared in 1) was reacted with 10U of aatII (Takara) for 2 hours at 37°C, followed by electrophoresis, and a pUC fmo+tnaA aatll vector was prepared through gel extraction.

Using the chromosomal DNA of wild-type Salmonella LT2 as a template, PCR was performed using the primer pairs in Table 2 and PrimeSTAR Max DNA Polymerase 2X master mix. PCR conditions were 98° C. 3 minutes and 35 cycles of 98° C. 10 seconds-55° C. 5 seconds-72° C. 2 minutes and 75° C. 3 minutes. The PCR product was subjected to electrophoresis, and a glmS insert was prepared by gel extraction from the electrophoretic gel.

100 ng of pUC fmo+tnaA aatll vector and 40 ng of glmS insert were reacted with 0.6 U of T4 DNA polymerase (NEB) at room temperature (25°C) for 2 minutes, and then reacted for 10 minutes in an ice water bath, and then transformed into DH5a competent cells. Converted. The transformed cells were plated on LB amp solid medium, and cultured at 37° C. having antibiotic resistance, and colonies having antibiotic resistance were selected. Among the selected colonies, 6 candidates were requested to Cosmogenetech to analyze the nucleotide sequence of the candidate, confirmed the insertion of the glmS gene, and established as pUC fmo+tnaA+glmS plasmid (FIG. 5).

3) Transformation of strain and assay for indirubin production

Using the pUC fmo+tnaA+glmS plasmid prepared in 2), aroA glmS- Salmonella strain, KST0650 glmS- Salmonella strain, ppGpp glmS- Salmonella strain, galE flhDC glmS- Salmonella strain was transformed, and smeared on LB amp medium. Then, it was cultured at 37° C. for 36 hours to test the production of indirubin. In addition, in Salmonella, indirubin is normally produced or not detected in a very small amount, whereas E. coli is reported to produce indirubin. To compare the indirubin-producing ability of the transformed Salmonella strain, E. coli M1655 glmS-strain was used in Chonnam National University. After being dispensed in the professor's laboratory and transformed together, the production of indirubin was tested for comparison.

6 is a photograph showing the results, aroA galE- Salmonella strain transformed with pBad18 strain as a control (control). As can be seen in Figure 6, ppGpp glmS- strain showed that it can generate indirubin weakly by transformation, but most of the indigo was formed. The M1655 glmS- strain, an E. coli strain, showed pink color to show that indirubin was produced, but the speed was slow and indigo was mixed. galE flhDC glmS- showed the fastest and clearest pink color, showing that indirubin production was more effective than E. coli. Although not shown in the drawing, aroA glmS- and KST0650 glmS- produced a small amount of indirubin, similar to ppGpp glmS-, and mostly indigo.

Accordingly, fmo+tnaA+glmS/galE flhDC glmS- was established as the optimal strain, and it was named fmo-tnaA-glmS/galE flhDC glmS Salmonella, and deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on March 20, 2020. (Accession No. KCTC18809P). 7 is a schematic diagram of the strain.

Figure 8 is an additional photograph showing the indirubin production of the strain, 1 is an untransformed galE flhDC glmS- strain, 2 is a galE flhDC glmS- strain transformed with fmo+tnaA+glmS plasmid in a solid LB amp medium It is a photograph of incubation at 37°C for 36 hours, and 3 is an enlarged photograph of 2. In the case of not being transformed with fmo+tnaA+glmS, indirubin is not produced, but it can be more clearly confirmed that indirubin is formed by transformation. The production of indirubin was further confirmed again by HPLC.

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Claims (7)

An anticancer attenuated Salmonella strain that is transformed with a plasmid containing fmo (flavin-containing monooxygenase) gene and tnaA (tryptophanase) gene to produce indirubin at a cancer site.
The method of claim 1,
The attenuated Salmonella strain has the glmS gene deleted,
The plasmid is an anticancer attenuated Salmonella strain, characterized in that it contains the glmS gene.
The method according to claim 1 or 2,
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, 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, flhDC, relA and spoT Anticancer attenuated Salmonella strain, characterized in that to induce loss.
The method of claim 3,
The attenuated Salmonella strain is an anticancer attenuated Salmonella strain, characterized in that the functions of the galE gene and the flhDC gene are lost.
The method of claim 4,
The strain is Salmonella strain, characterized in that the accession number KCTC18809P.
An anticancer composition containing the Salmonella strain of claim 1 or 2 as an active ingredient.
The method of claim 6,
The Salmonella strain is an anticancer composition, characterized in that the accession number KCTC18809P.

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