CN112190717B

CN112190717B - Application of attenuated salmonella typhimurium and genetically engineered bacteria thereof in preparation of medicines for treating acute leukemia

Info

Publication number: CN112190717B
Application number: CN202011111337.1A
Authority: CN
Inventors: 赵子建; 李芳红; 李美蓉; 赵正刚; 周素瑾; 赖运浩
Original assignee: Guangzhou Huajin Pharmaceutical Technology Co ltd
Current assignee: Guangzhou Huajin Pharmaceutical Technology Co ltd
Priority date: 2020-10-16
Filing date: 2020-10-16
Publication date: 2023-12-15
Anticipated expiration: 2040-10-16
Also published as: CN112190717A

Abstract

The invention discloses an attenuated salmonella typhimurium and application of genetically engineered bacteria thereof in preparing a medicament for treating acute leukemia, and relates to the technical field of medicaments. The genetically engineered bacterium carries recombinant expression plasmid cloned with L-methioninase (L-methioninase or methioninase) gene, can continuously express the L-methioninase in tumor tissues, consumes a large amount of methionine and other nutrients, ensures that tumor cells lack nutrition and grow slowly, has obvious growth inhibition effect on acute leukemia cells, and can be used for preparing medicines for treating acute leukemia.

Description

Application of attenuated salmonella typhimurium and genetically engineered bacteria thereof in preparation of medicines for treating acute leukemia

Technical Field

The invention relates to the technical field of medicines, in particular to an attenuated salmonella typhimurium and application of genetically engineered bacteria thereof in preparation of medicines for treating acute leukemia.

Background

The global disease burden institute estimated that the total number of global leukemia cases increased by 26% from 2005 to 2015. In 2012, more than 35 ten thousand leukemia cases are newly increased worldwide, and 26.5 ten thousand leukemia death cases are newly increased; in 2018, leukemia causes 437033 cancer cases and 309006 cancer deaths, the incidence rates of men and women are 6.1/10 ten thousand and 4.3/10 ten thousand respectively, and the mortality rates of men and women are 4.2/10 ten thousand and 2.8/10 ten thousand respectively, which are the 15 th most common cancers worldwide and are also the main causes of the 11 th cancer deaths. Since 2006, leukemia incidence has increased by 0.6% annually on average, while mortality has decreased by 1.5% annually on average, accounting for 4% of total cancer deaths. Children and adolescents are the major populations with high incidence of leukemia and death, 29% of cancer-bearing deaths are the death from leukemia, with acute leukemia being the most dangerous and most common malignancy leading to childhood death. Acute leukemias include acute leukemia or acute lymphoblastic leukemia (Acute lymphocytic leukemia, ALL) and acute myelogenous leukemia or acute myelogenous leukemia (Acute myeloid leukemia, AML), which are rapid in onset, resistant to drugs, easy to relapse, more refractory to relapse and high in mortality, are the most common types of diseases in which leukemias die, and are one of the most common cancers with high morbidity and mortality in children and adolescents.

With the continuous improvement of drugs and risk classification management in recent decades, acute leukemia treatment for children has advanced tremendously, however, adult and especially aged acute leukemia patients have poor prognosis, many patients fail treatment after relapse, and eventually die. At present, the treatment of acute leukemia is mainly chemotherapy, and the main chemotherapy scheme is to treat with high-dose cytarabine and anthracycline chemical drugs. Only when chemotherapy fails or the disease recurs, bone marrow transplantation or hematopoietic stem cell transplantation is considered. However, chemotherapy has strong side effects and drug resistance is easy to relapse, often resulting in treatment failure and death. Although bone marrow or hematopoietic stem cell transplantation is effective, it also has problems of difficult donor search, high operation cost, difficult pain and tolerance in the operation process, and a certain recurrence risk. Therefore, the search for new drugs with small side effects, reliable curative effect and economy has great significance for treating acute leukemia, especially recurrent acute leukemia.

Clinical reports of spontaneous remissions obtained after severe bacterial infections in a number of acute leukemia patients, combined with the current very high-attention oncological bacterial therapies, suggest that bacterial therapies may be a new direction for the treatment of acute leukemia. Among a variety of bacteria used in tumor therapy research, salmonella is widely used in the treatment of various solid tumors due to its excellent tumor targeting and anti-tumor effects. Salmonella is a population of gram-negative, invasive intracellular facultative anaerobes that are parasitic in the human and animal gut. VNP20009 is an attenuated Salmonella typhimurium strain with msbB and pur I genes deleted, and is genetically stable and sensitive to antibiotics. The msbB gene is necessary for acylation of lipid to endotoxin, and deletion of the msbB gene can not be acylated at the end of lipid A, so that toxicity is reduced; pur I gene is involved in purine metabolism, and the deletion thereof makes exogenous adenine necessary for bacterial reproduction. VNP20009 also reduced tumor necrosis factor (tumor necrosis factor, TNF) produced by the self-induced body, thereby reducing inflammatory responses. The endotoxin toxicity of the genetically engineered attenuated salmonella VNP20009 is reduced by 5-ten thousand times, and its tolerance and safety have been confirmed in a first clinical trial. VNP20009 has been widely used in cancer research and can act on a variety of mouse solid tumor models including melanoma, lung cancer, colon cancer, breast cancer, and renal cancer. VNP20009 can also be highly targeted to aggregate on tumor sites, and can be used as a tumor gene therapy vector. Researchers found that the number of VNP20009 in tumors is 200-l 000 times higher than that in main organs such as liver in mouse models of various solid tumors. The VNP20009 can be preferentially aggregated and propagated in the hypoxic necrotic area of the tumor tissue, and the amplification times of bacteria in the tumor tissue are obviously higher than those of normal tissue within the same time, so that the attenuated salmonella becomes a novel anti-tumor preparation and a carrier for tumor targeted therapy. Possible mechanisms by which salmonella causes a reduction in tumor growth include: the nutrients required for tumor growth are consumed by bacteria, and enzymes produced by the bacteria, such as asparaginase, can deplete essential amino acids for tumor growth; local toxins secreted by bacteria to the extracellular microenvironment or produced tumor necrosis factor alpha can affect tumor angiogenesis; in addition, nonspecific inflammatory responses at the bacterial growth site can potentially activate anti-tumor T cells. Our earlier study found that VNP20009 not only induced and inhibited apoptosis of acute leukemia cells, inhibited growth of subcutaneous transplants, but also activated various cytokines and immune cell-mediated immune responses of mice to exert antitumor activity.

In order to maintain a high proliferation rate of tumor cells, sufficient nutrition is required, and in addition to sugar, methionine (Met), glutamine, arginine, and the like are required in particularly large amounts. Studies have demonstrated that Met dependence is a common feature of most tumor cells, such as breast, lung, colon, kidney, bladder, melanoma, glioma, etc., whereas normal cells are free of Met dependence. Several in vitro and in vivo experiments have successively demonstrated that direct consumption of methionine deficient diets can delay proliferation of tumor cells. However, if Met is absent or insufficient in diet for a long period of time, malnutrition and metabolic disorder of the organism can be caused, and canceration can be aggravated due to the fact that DNA is in hypomethylation state for a long period of time. Then, met is specifically decomposed by methioninase (L-methioninase) to lower methionine level in vivo, and thus tumor cell growth can be more effectively inhibited or resolved. Animal experiments have shown that intraperitoneal injection of methioninase can inhibit the growth of Jitian sarcoma and lung tumor in nude mice. Clinical trials four patients with breast, lung, kidney and lymphoma cancer respectively were given an intravenous methioninase every 24h, which was found to significantly reduce the methionine content in the plasma. However, since the mammal itself does not express methioninase, the exogenous administration mode has a certain side effect and often causes an immune response of the body.

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of attenuated salmonella typhimurium and genetically engineered bacteria thereof in preparing biological medicaments for treating acute leukemia.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention provides application of attenuated salmonella typhimurium in preparation of a medicament for treating acute leukemia, wherein the attenuated salmonella typhimurium is attenuated salmonella typhimurium VNP20009.

The invention provides application of genetically engineered bacteria in preparation of medicines for treating acute leukemia, wherein the genetically engineered bacteria are attenuated salmonella typhimurium VNP20009 carrying plasmids, and are named as VNP20009-M.

Wherein the plasmid is pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid. The plasmid was transferred into attenuated salmonella typhimurium VNP20009 by electrotransformation. The electric conversion condition is voltage 2400V, resistance 400 omega, capacitance 25 muF, and discharge time 4ms.

The invention also provides application of the genetically engineered bacterium in preparing a medicament for treating acute leukemia, wherein the genetically engineered bacterium is attenuated salmonella typhimurium VNP20009 carrying a plasmid, and the plasmid is cloned with an L-methioninase gene.

The plasmid is pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid. The construction method of the genetically engineered bacterium comprises the following steps: subcloning the L-methioninase gene into plasmid to obtain L-methioninase expression plasmid, and electrically transforming the L-methioninase expression plasmid into attenuated Salmonella typhimurium VNP20009. The electric conversion condition is voltage 2400V, resistance 400 omega, capacitance 25 muF, and discharge time 4ms.

Most preferably, in the process of constructing the genetically engineered bacterium, when the pSVSPORT plasmid is selected, the L-methioninase gene is subcloned into the plasmid through Kpn I and Hind III cleavage sites to obtain an L-methioninase expression plasmid, and then the L-methioninase expression plasmid is electrically transformed into attenuated Salmonella typhimurium VNP20009 to obtain the genetically engineered bacterium.

The administration mode of the attenuated salmonella typhimurium and the genetically engineered bacteria is preferably intratumoral injection or intravenous injection.

The beneficial effects are that: compared with the existing medicines, the microbial medicine for treating the acute leukemia takes the attenuated salmonella typhimurium VNP20009 as a biological carrier, and can efficiently and continuously express the L-methioninase in tumor tissues by utilizing a genetic engineering technology, so that methionine and other nutrients are consumed in a quantity, the tumor cells lack nutrition and grow slowly, and the microbial medicine has an obvious inhibition effect on the acute leukemia cells; the preparation method has the advantages of no toxic or side effect, good safety, simple preparation method, easy operation, large-scale production and low cost, and can be used for preparing the medicine for treating the acute leukemia.

Drawings

FIG. 1 is a diagram showing the double digestion identification of 1% agarose gel by plasmid pSVSPORT-L-methioninase.

FIG. 2 is a graph showing the results of Western blot identification of L-methioninase expression.

FIG. 3 is a graph showing the results of detecting the activity of L-methioninase in Salmonella.

FIG. 4 is a graph showing the effect of intratumoral injection of VNP20009-M on the body weight of L1210 tumor-bearing nude mice.

FIG. 5 is a graph of the volume growth of L1210 subcutaneous grafts following intratumoral injection of VNP20009-M.

FIG. 6 is a graph showing the results of the relative inhibition of tumor volume growth of L1210 subcutaneous grafts after intratumoral injection of VNP20009-M.

FIG. 7 is a graph of the size results of HL-60 subcutaneous transplants at the end of intratumoral injection of VNP20009-M treatment.

FIG. 8 is a weight map of HL-60 subcutaneous engraftment tumor at the end of intratumoral injection VNP20009-M treatment.

FIG. 9 is a graph showing HE staining results of L1210 subcutaneous transplanted tumor tissue at the end of intratumoral injection of VNP20009-M treatment.

FIG. 10 is a graph showing the effect of intratumoral injection of VNP20009-M on the body weight of HL-60 tumor-bearing nude mice.

FIG. 11 is a graph of the volume growth of HL-60 subcutaneous grafts following intratumoral injection of VNP20009-M.

FIG. 12 is a graph showing the results of the relative inhibition of tumor volume growth of HL-60 subcutaneous grafts after intratumoral injection of VNP20009-M.

FIG. 13 is a graph of the size results of HL-60 subcutaneous transplants at the end of intratumoral injection of VNP20009-M treatment.

FIG. 14 is a weight map of HL-60 subcutaneous engraftment tumor at the end of intratumoral injection VNP20009-M treatment.

FIG. 15 is a graph showing HE staining results of HL-60 subcutaneous transplanted tumor tissue at the end of intratumoral injection of VNP20009-M treatment.

FIG. 16 is a graph showing the effect of VNP20009-M on body weight of human MLL-AF 9-driven systemic AML mice.

FIG. 17 is a schematic diagram showing in vivo GFP inhibition by VNP20009-M in tail vein injection mice ⁺ Results of proliferation of human MLL-AF 9-driven systemic AML leukemia cellsA drawing.

FIG. 18 Tail vein injection of VNP20009-M in vivo GFP in mice ⁺ Results of the relative inhibition of proliferation of human MLL-AF 9-driven systemic AML leukemia cells.

Detailed Description

The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.

Example 1: construction of genetically engineered bacteria.

(1) A plasmid expressing the L-methioninase gene was constructed.

The L-methioninase (GenBank: L43133.1) gene was synthesized first subcloned into pUC57 plasmid (Kirschner Co.), and then subcloned into pSVSPORT plasmid (Invitrogen) through KpnI and HindIII cleavage sites, resulting in pSVSPORT-L-methioninase expression plasmid. The specific construction process is as follows:

the pSVSPORT plasmid was digested with KpnI and HindIII in the following system: 2. Mu.g of plasmid DNA, 3. Mu.L of 10 Xbuffer, 1.5. Mu.L of KpnI enzyme, 1.5. Mu.L of HindIII enzyme, and ddH were added ₂ O was made up to 30. Mu.L and incubated at 37℃for 3h. The cleavage system was then separated by electrophoresis in a 1% agarose gel, a 4.1kb sized DNA band was excised and the DNA was purified using a gel recovery purification kit.

The DNA fragment of the coding region of L-methioninase obtained by total gene synthesis was subcloned into pUC57 plasmid (Kirschner Co.) and digested with Kpn I and Hind III in the following system: mu.g of plasmid DNA, 3. Mu.L of 10 XBuffer, 1.5. Mu.L of KpnI enzyme, 1.5. Mu.L of HindIII enzyme, and ddH were added ₂ O was made up to 30. Mu.L and incubated at 37℃for 3h. The cleavage system was then separated by electrophoresis in a 1% agarose gel, a 1.2kb sized DNA band was excised and the DNA was purified using a gel recovery purification kit.

pSVSPORT (Kpn I/Hind III) was ligated to the L-methinase encoding region DNA fragment (Kpn I/Hind III) and 2. Mu.L vector, 6. Mu.L insert, 1. Mu. L T4 DNA ligase were added to the ligation reaction and incubated for 16h at 16 ℃.

The ligation product was transformed into competent cells of E.coli DH 5. Alpha (Takara). Taking a tube of 50 mu L DH5 alpha competent cells, putting the tube on ice, adding 5 mu L of the connecting product into the tube after the DH5 alpha competent cells are melted, flicking and mixing the mixture uniformly, and incubating the mixture on ice for 30min; heat-shock at 42 ℃ for 60s, and standing on ice for 2min; adding 500 mu L of non-resistant LB liquid medium, and shake culturing at 37 ℃ for 1h; centrifuging at 4000rpm for 5min, sucking away, reserving about 100 mu L of culture medium, blowing the bacterial sediment uniformly by a pipette, and coating on an ampicillin-resistant LB culture medium plate. The plates were then incubated at 37℃for 16h.

After the cloning, the monoclonal colony is picked up into 3mL LB culture solution containing ampicillin, shake cultured for 16h at 37 ℃, plasmid DNA is extracted, and is identified by Kpn I and Hind III digestion, and two DNA bands of 4.1kb and 1.2kb can be obtained in positive cloning, as shown in FIG. 1. The sequence of the positive clone was further determined to be perfectly correct by sequencing.

(2) Constructing salmonella genetic engineering bacteria carrying empty load expression plasmids and salmonella genetic engineering bacteria carrying recombinant expression plasmids cloned with genes of L-methioninase.

The empty vector pSVSPORT and the recombinant expression plasmid pSVSPORT-L-methioninase were respectively electrotransformed into VNP20009 strain (YS 1646, ATCC No. 202165) to construct a Salmonella genetically engineered bacterium, and designated as VNP20009-V and VNP20009-M, respectively. The specific construction process is as follows:

competent bacteria VNP20009 was placed on ice, transferred into a pre-chilled electric rotor after melting, 2. Mu.L plasmid was added thereto, gently flicked, mixed and incubated on ice for 1min. The electric rotating cup is placed in an electric rotating instrument, and the conditions are set to be 2400V, 400 omega of resistance, 25 mu F of capacitance and 4ms of discharge time. Immediately after the electric shock, 1mL of SOC medium was added and gently mixed. Shake culturing at 37deg.C for 1 hr; centrifuging at 4000rpm for 5min, sucking away, reserving about 100 mu L of culture medium, blowing the bacterial sediment uniformly by a pipette, and then coating on an ampicillin-resistant LB-O medium plate. The plates were then incubated at 37℃for 16h. After VNP20009-V and VNP20009-M were cultured with LB-O, the plasmids were extracted and the restriction enzyme was identified correctly.

Taking 1×10 ⁸ Protein cleavage for salmonellaExtracting protein from the solution, performing 10% SDS-PAGE electrophoresis, transferring to PVDF membrane by pressure stabilizing ice bath, blocking with BSA at room temperature for 1h, rinsing with TBST for 3×5min, adding rabbit anti-L-methioninase antibody (1:1000), and incubating overnight at 4deg.C. TBST was rinsed 3 times, each time for 5min, HRP-labeled anti-rabbit secondary antibody (1:10000) was added, incubated at room temperature for 1h, TBST was rinsed 3 times, each time for 5min, and developed by ECL chemiluminescence. As a result, as shown in FIG. 2, a specific band was present at a molecular weight of about 43kD, indicating that VNP20009-M significantly increased the expression level of L-methioninase as compared with VNP20009 and VNP 20009-V.

L-methionine and pyridoxal were mixed with VNP20009, VNP20009-V and VNP20009-M cells, respectively, incubated at 37℃for 10min, stopped with 50% trichloroacetic acid, the supernatant was centrifuged, and after incubation at 50℃for 30min, the absorbance at 320nm was determined, the amount of enzyme that catalytically converted 1. Mu. Mol of alpha-ketobutyric acid per minute was defined as 1 enzyme activity unit, after thorough mixing with 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH). The results showed (FIG. 3) that the methioninase activity of Salmonella VNP20009-M was 10-fold higher than that of VNP20009 and VNP 20009-V.

Example 2: anti-tumor effect of genetically engineered bacteria VNP20009-M on L1210 subcutaneous transplantation tumor.

1. The mice L1210 acute T lymphocyte leukemia cells were cultured in DMEM medium containing 10% fetal bovine serum, respectively, in a cell number of 1X 10 ⁵ The individual was inoculated subcutaneously into the right underarm of nude mice. Every 2 to 3 days, the state of the mice was observed, and the tumor size was measured with a vernier caliper (volume=0.5×length×width ² ). When the tumor volume reaches 80-100 mm ³ At this time, tumor-bearing nude mice were randomly grouped: PBS group and VNP20009-M group.

2. VNP20009-M was cultured with LB-O, when OD.apprxeq.0.6, the cells were collected and then resuspended with PBS at 2X 10 ⁶ CFU/dose alone, was administered by intratumoral injection and the control group was injected with equal volume PBS. Following dosing, the nude mice were observed for activity, feeding, and body weight. The results are shown in FIG. 4, where the body weight of the mice was unaffected after bacterial injection. Furthermore, the feeding and the feces of the nude mice are also not abnormal, which indicates that VNP20009-M has no obvious toxicity to the nude mice.

3. Every 2-3d, the length and width of the tumor were measured, the tumor volume was calculated, and the tumor volume change curve (fig. 5) and the relative proliferation curve (fig. 6) of the nude mice were plotted. The results show that the L1210 tumor grows rapidly, the tumor volume of the bacteria-administered treatment group is significantly smaller than that of the control group, and the relative proliferation rate of the tumor is significantly reduced. Mice were sacrificed at the end of treatment, subcutaneous tumors were dissected, photographed (fig. 7), and weighed (fig. 8). The result shows that after the salmonella genetically engineered bacteria VNP20009-M is administered, the tumor growth is slow, and the volume and the weight are obviously lower than those of a PBS group. The results show that VNP20009-M can significantly inhibit the growth of L1210 graft tumor.

4. Tumor tissues were also fixed overnight with 4% formalin and sectioned by paraffin embedding for hematoxylin-eosin (HE) staining. Comparing with PBS control group, pathological section HE staining (figure 9) shows that the tumor after VNP20009-M injection generates large-area tissue necrosis, and has obvious inhibition effect on the growth of L1210 subcutaneous transplantation tumor.

Example 3: the genetically engineered bacterium VNP20009-M has anti-tumor effect on HL-60 subcutaneous transplantation tumor.

1. Human HL-60 acute myelogenous leukemia cells were cultured in IMDM medium containing 10% fetal bovine serum, respectively, at a cell number of 5×10 ⁶ The individual was inoculated subcutaneously into the right underarm of nude mice. Every 2 to 3 days, the state of the mice was observed, and the tumor size was measured with a vernier caliper (volume=0.5×length×width ² ). When the tumor volume reaches 80-100 mm ³ At this time, tumor-bearing nude mice were randomly grouped: PBS group and VNP20009-M group.

2. VNP20009-M was cultured with LB-O, when OD.apprxeq.0.6, the cells were collected and then resuspended with PBS at 2X 10 ⁶ CFU/dose alone, was administered by intratumoral injection and the control group was injected with equal volume PBS. Following dosing, the nude mice were observed for activity, feeding, and body weight. The results are shown in fig. 10, and the body weight of the mice was not affected after the bacteria injection. Furthermore, the feeding and the feces of the nude mice are also not abnormal, which indicates that VNP20009-M has no obvious toxicity to the nude mice.

3. Every 2-3d, the length and width of the tumor were measured, the tumor volume was calculated, and the tumor volume change curve (fig. 11) and the relative proliferation curve (fig. 12) of the nude mice were plotted. The results show that the tumor volume of the bacteria-administered treatment group is significantly smaller than that of the control group, and the relative proliferation rate of the tumor is significantly reduced. Mice were sacrificed at the end of treatment, subcutaneous tumors were dissected, photographed (fig. 13), and weighed (fig. 14). The result shows that after the salmonella genetically engineered bacteria VNP20009-M is administered, the tumor growth is slow, and the volume and the weight are obviously lower than those of a PBS group. The results show that VNP20009-M can significantly inhibit the growth of HL-60 graft tumor.

4. Tumor tissues were also fixed overnight with 4% formalin and sectioned by paraffin embedding for hematoxylin-eosin (HE) staining. Comparing with PBS control group, pathological section HE staining (figure 15) shows that tumor after VNP20009-M injection generates large-area tissue necrosis, and has remarkable inhibition effect on the growth of HL-60 subcutaneous transplantation tumor.

Example 4: anti-tumor effect of genetically engineered bacteria VNP20009-M on MLL-AF 9-driven AML mice.

1. MLL-AF9 driven AML cell (with GFP reporter gene) freezing tube is taken out from liquid nitrogen tank, then is quickly put into water bath at 37 ℃ for resuscitation, washed 2 times by sterile PBS, and then is subjected to trypan blue staining counting, and the cell concentration is regulated to 1X 10 ⁶ after/mL, 200. Mu.L of the cell fluid was withdrawn by 1X 10 using a 1mL syringe ⁶ AML leukemia cells are treated by mouse tail vein injection to C57BL/6J male mice to induce morbidity;

2. the following day, mice were randomly divided into PBS group and VNP20009-M group; VNP20009-M was cultured with LB-O, when OD.apprxeq.0.6, the cells were collected and then resuspended with PBS at 2X 10 ⁶ The CFU/dose is administered by tail vein injection, and the control group is injected with PBS with the same volume; after one week, the administration was again performed as described above.

3. After self-administration, the drinking water diet and activity of the mice are observed and recorded every day, and the survival condition is recorded. The results are shown in fig. 16, and the body weight of the mice was not affected after the bacteria injection. And the mice have no abnormal food and feces, which indicates that VNP20009-M has no obvious toxicity to the mice.

4. After the red blood cells are lysed by weekly blood sampling, GFP in the peripheral blood leukocytes of mice is detected by flow cytometry ⁺ Leukemia cell content, i.e. detection of GFP ⁺ % of GFP (FIG. 17) and the control group ⁺ % is divided intoThe parent calculates the relative inhibition rate (fig. 18). The results show that the VNP20009-M tail vein can obviously reduce GFP in peripheral blood leucocytes of mice after administration ⁺ The leukemia cell content of (2) can inhibit proliferation of the mice, and shows that VNP20009-M has a remarkable anti-tumor effect on MLL-AF 9-driven AML mice.

The invention shows that the attenuated salmonella typhimurium gene engineering bacteria VNP20009-M with recombinant expression plasmid capable of cloning and expressing L-methioninase gene can continuously express L-methioninase in tumor tissues, consume a large amount of methionine and other nutrients, ensure that tumor cells lack nutrition and grow slowly, have obvious inhibition effects on acute leukemia cell subcutaneous transplantation tumor and MLL-AF9 driving AML, and can be used for preparing medicines for treating acute leukemia. The above expression vector plasmids are not limited to pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid, and other plasmids cloned with L-methioninase gene have similar effects.

Claims

1. The application of the genetically engineered bacterium in preparing a medicament for treating acute leukemia is characterized in that the genetically engineered bacterium is attenuated salmonella typhimurium VNP20009 carrying a plasmid, wherein the plasmid is cloned with an L-methioninase gene.

2. The use according to claim 1, wherein the plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcdna3.1 plasmid, a pBR322 plasmid or a pET23a plasmid.

3. The use according to claim 1 or 2, wherein the construction method of the genetically engineered bacterium comprises: subcloning the L-methioninase gene into plasmid to obtain L-methioninase expression plasmid, and electrically transforming the L-methioninase expression plasmid into attenuated Salmonella typhimurium VNP20009.

4. The method according to claim 3, wherein the electrical conversion conditions are 2400V, 400 Ω, 25 μF capacitance, and 4ms discharge time.

5. The use according to any one of claims 1 to 4, wherein the administration is intravenous.