CN112190717A

CN112190717A - Attenuated salmonella typhimurium and application of genetically engineered bacterium thereof in preparation of medicine for treating acute leukemia

Info

Publication number: CN112190717A
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: Guangdong University of Technology
Current assignee: Guangzhou Huajin Pharmaceutical Technology Co ltd
Priority date: 2020-10-16
Filing date: 2020-10-16
Publication date: 2021-01-08
Anticipated expiration: 2040-10-16
Also published as: CN112190717B

Abstract

The invention discloses an application of attenuated salmonella typhimurium and genetically engineered bacteria thereof in preparing a medicament for treating acute leukemia, and relates to the technical field of medicaments. The gene engineering bacteria carries recombinant expression plasmid with cloned L-methioninase gene, and can express L-methioninase continuously in tumor tissue, consume methionine and other nutrients greatly, make tumor cell lack nutrition, grow slowly, and have obvious growth inhibiting effect on acute leukemia cell, so that it can be used for preparing medicine for treating acute leukemia.

Description

Attenuated salmonella typhimurium and application of genetically engineered bacterium thereof in preparation of medicine for treating acute leukemia

Technical Field

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

Background

Global disease burden institute estimates that the total number of leukemia cases worldwide increases by 26% from 2005 to 2015. In 2012, more than 35 ten thousand of new leukemia cases and 26.5 ten thousand of death cases of leukemia are added worldwide; in 2018, the leukemia causes 437033 cancer cases and 309006 cancer deaths together, the morbidity of men and women is 6.1/10 ten thousand and 4.3/10 ten thousand respectively, and the mortality of men and women is 4.2/10 ten thousand and 2.8/10 ten thousand respectively, which are the most common cancer at the 15 th position in the world and are also the main reason of the 11 th cancer death. Since 2006, the incidence of leukemia has increased by an average of 0.6% per year, while the mortality has decreased by an average of 1.5% per year, accounting for 4% of total cancer deaths. Children and adolescents are the main population with high incidence and death of leukemia, and 29% of cancer-deaths are deaths from leukemia, with acute leukemia being the most harmful, the most common malignancy that causes death in children. Acute leukemias include Acute lymphoblastic or Acute Lymphocytic Leukemia (ALL) and Acute myeloid or Acute Myelogenous Leukemia (AML), which are rapidly onset, resistant, easily relapsed, more difficult to treat after relapse, and high in mortality, are the most common types of leukemic death, and are among the most common cancers with high morbidity and mortality in children and adolescents.

With the continuous improvement of drug and risk classification management in recent decades, the treatment of acute leukemia in children is greatly improved, however, the prognosis of adult acute leukemia patients, especially the elderly acute leukemia patients is poor, and many patients fail to treat after relapse and finally die. Chemotherapy is still the main treatment of acute leukemia at present, and the main chemotherapy scheme is the treatment with large dose of cytarabine and anthracycline chemical drugs. Bone marrow transplantation or hematopoietic stem cell transplantation is considered only when chemotherapy fails or the disease recurs. However, chemotherapy has strong side effects and resistance to drugs is susceptible to relapse, often resulting in treatment failure and death. Although the effect of bone marrow or hematopoietic stem cell transplantation is good, the problems of difficult donor search, high operation cost, difficult pain and tolerance in the operation process and the like exist, and a certain recurrence risk still exists. Therefore, the search for new economic medicines with small side effect and reliable curative effect has great significance for treating acute leukemia, especially recurrent acute leukemia.

The clinical report that a plurality of acute leukemia patients obtain spontaneous remission after serious bacterial infection is combined with the current tumor bacterial therapy with high attention, and the bacterial therapy is suggested to be a new direction for treating acute leukemia. Among various bacteria used for tumor treatment research, salmonella is widely applied to various solid tumor treatments because of its good tumor targeting and anti-tumor effects. Salmonella is a group of gram-negative, invasive, intracellular facultative anaerobes that parasitize in the gut of humans and animals. VNP20009 is an attenuated salmonella typhimurium strain with msbB and pur I gene deletion, and is stable in heredity and sensitive to antibiotics. The msbB gene is necessary for the acylation of lipid to endotoxin, and the deletion of the msbB gene prevents the acylation of the tail end of the lipid A, thereby reducing the toxicity; the pur I gene is involved in purine metabolism, and its deletion requires exogenous gonadal purine for bacterial reproduction. VNP20009 also reduces Tumor Necrosis Factor (TNF) produced by the auto-induced body, thereby reducing the inflammatory response. The toxicity of endotoxin generated by genetically engineered attenuated salmonella VNP20009 is reduced by 5 ten thousand times, and the tolerance and safety of the attenuated salmonella are proved in a first-stage clinical test. VNP20009 has been widely used in cancer research and it can act on a variety of mouse solid tumor models, including melanoma, lung, colon, breast, kidney. VNP20009 can also highly target and gather at tumor site, and can be used as tumor gene therapy vector. Researchers found that the number of VNP20009 in tumors was 200-to l 000-fold higher than that in major organs such as liver in mouse models of various solid tumors. VNP20009 can be preferentially gathered and propagated in the hypoxic necrosis area of tumor tissue, and the amplification times of bacteria in the tumor tissue are obviously higher than those of normal tissue in the same time, so that attenuated salmonella becomes a novel anti-tumor preparation and a carrier for tumor targeted therapy. Possible mechanisms by which salmonella cause a reduction in tumor growth include: nutrients required for tumor growth are consumed by bacteria, and enzymes produced by the bacteria, such as asparaginase, can exhaust amino acids essential for tumor growth; local toxins secreted by bacteria into the extracellular microenvironment or produced tumor necrosis factor alpha can affect tumor angiogenesis; in addition, non-specific inflammatory reactions at the site of bacterial growth can potentially activate anti-tumor T cells. Earlier researches show that VNP20009 can not only induce and inhibit apoptosis of acute leukemia cells and inhibit growth of subcutaneous transplanted tumors, but also activate various cytokines and immune cell-mediated immune reactions of mice to play an anti-tumor activity.

Tumor cells require sufficient nutrients to maintain their high proliferation rate, and in addition to sugars, Methionine (Met, also referred to as Methionine), glutamine, arginine, and the like are particularly required in large amounts. Studies have shown that Met dependence is a common feature of most tumor cells, such as breast, lung, colon, kidney, bladder, melanoma, glioma, etc., while normal cells do not have Met dependence. Several in vivo and in vitro experiments have successively demonstrated that the direct consumption of methionine-deficient diets can delay the proliferation of tumor cells. However, long-term deficiency or insufficiency of Met in the diet can cause malnutrition and metabolic disturbance of the body, and can also aggravate canceration due to the fact that DNA is in a hypomethylation state for a long time. Then, by specifically decomposing Met by methioninase (L-methioninase), thereby reducing methionine levels in vivo, tumor cell growth can be more effectively inhibited or regressed. Animal experiments have demonstrated that intraperitoneal injection of methioninase can inhibit the growth of sarcoma of gytian and lung tumors in nude mice. Clinical trials have shown that methioninase can significantly reduce the methionine content in plasma by intravenous injection of methioninase every 24h for four patients with breast, lung, kidney and lymphoma tumors, respectively. However, since mammals do not express methioninase, exogenous administration has a certain side effect and often causes immune reaction in the body.

Disclosure of Invention

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

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention provides application of attenuated salmonella typhimurium in preparation of a medicine for treating acute leukemia, wherein the attenuated salmonella typhimurium is attenuated salmonella typhimurium VNP 20009.

The invention provides an application of genetically engineered bacterium in preparation of a medicament for treating acute leukemia, wherein the genetically engineered bacterium is attenuated salmonella typhimurium VNP20009 carrying plasmids and is named VNP 20009-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 electrotransformation conditions are voltage 2400V, resistance 400 omega, capacitance 25 muF and discharge time 4 ms.

The invention also provides an 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 plasmids, and L-methioninase genes are cloned on the plasmids.

The plasmid is pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid. The construction method of the genetic engineering bacteria comprises the following steps: subcloning the L-methioninase gene into a plasmid to obtain an L-methioninase expression plasmid, and electrically transforming the L-methioninase expression plasmid into the attenuated Salmonella typhimurium VNP 20009. The electrotransformation conditions are voltage 2400V, resistance 400 omega, capacitance 25 muF and discharge time 4 ms.

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 the 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.

Has the advantages that: compared with the existing medicines, the microbial medicine for treating acute leukemia of the invention takes attenuated salmonella typhimurium VNP20009 as a biological carrier, can efficiently and continuously express L-methioninase in tumor tissues by utilizing a genetic engineering technology, consumes methionine and other nutrient substances in quantity, ensures that tumor cells lack nutrition and grow slowly, and has obvious inhibiting effect on the acute leukemia cells; the traditional Chinese medicine composition 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 medicines for treating acute leukemia.

Drawings

FIG. 1 is a diagram of a double-restriction and identification 1% agarose gel electrophoresis of plasmid pSVSPORT-L-methioninase.

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

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

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

FIG. 5 is a graph of L1210 subcutaneous graft tumor volume growth following intratumoral injection of VNP 20009-M.

FIG. 6 is a graph showing the results of the volume growth relative inhibition rate of L1210 subcutaneous grafts after intratumoral injection of VNP 20009-M.

FIG. 7 is a graph of the size results of terminal HL-60 subcutaneous transplantable tumors treated with intratumoral VNP 20009-M.

FIG. 8 is a weight plot of the end stage of intratumoral injection of VNP20009-M for HL-60 subcutaneous transplantable tumors.

FIG. 9 is a graph showing HE staining results of L1210 subcutaneous transplanted tumor tissue at the end of intratumoral injection of VNP 20009-M.

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 HL-60 subcutaneous graft tumor volume growth after intratumoral injection of VNP 20009-M.

FIG. 12 is a graph showing the results of the volume growth relative inhibition rate of HL-60 subcutaneous transplanted tumors after intratumoral injection of VNP 20009-M.

FIG. 13 is a graph of the size results of an end-stage HL-60 subcutaneous transplanted tumor treated with intratumoral VNP 20009-M.

FIG. 14 is a weight plot of the end stage of intratumoral injection of VNP20009-M for HL-60 subcutaneous transplantable tumors.

FIG. 15 is a graph showing HE staining results of HL-60 subcutaneous transplanted tumor tissue at the end of the intratumoral injection of VNP 20009-M.

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

FIG. 17 is tail vein injection of VNP20009-M for inhibition of GFP in mice⁺Results of proliferation of human MLL-AF 9-driven systemic AML leukemia cells.

FIG. 18 is tail vein injection of VNP20009-M vs. in vivo GFP in mice⁺Results of the relative inhibition rate of cell proliferation of human MLL-AF 9-driven systemic AML leukemia.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: and (3) construction of the genetically engineered bacteria.

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

The L-methioninase (GenBank: L43133.1) gene was synthesized and subcloned into pUC57 plasmid (Kinsley), and then into pSVSPORT plasmid (Invitrogen) through Kpn I and Hind III cleavage sites to obtain pSVSPORT-L-methioninase expression plasmid. The specific construction process is as follows:

the pSVSPORT plasmid is double-digested by Kpn I and Hind III, and the digestion system is as follows: mu.g of plasmid DNA, 3. mu.L of 10 XBuffer, 1.5. mu.L of Kpn I enzyme, 1.5. mu.L of Hind III enzyme, ddH was added₂The volume is made up to 30 μ L by O and the bath is carried out at 37 ℃ for 3 h. The digested system was then separated by electrophoresis on a 1% agarose gel, a 4.1kb DNA band was excised, and the DNA was purified using a gel recovery purification kit.

Obtaining DNA fragment of L-methioninase coding region by whole gene synthesisSubcloning into pUC57 plasmid (Kisry) and double digestion with Kpn I and Hind III according to the following protocol: mu.g plasmid DNA, 3. mu.L 10 XBuffer, 1.5. mu.L Kpn I enzyme, 1.5. mu.L Hind III enzyme, ddH was added₂The volume is made up to 30 μ L by O and the bath is carried out at 37 ℃ for 3 h. The digested system was then separated by electrophoresis on a 1% agarose gel, a DNA band of 1.2kb in size was excised, and the DNA was purified using a gel recovery purification kit.

pSVSPORT (Kpn I/Hind III) and L-methioninase coding region DNA fragment (Kpn I/Hind III) were ligated by adding 2. mu.L of vector, 6. mu.L of insert, 1. mu. L T4 DNA ligase, and 16 ℃ by incubation for 16 h.

The ligation product was transformed into competent cells of e.coli DH5 α (Takara). Placing 50 μ L DH5 α competent cell on ice, adding 5 μ L ligation product after thawing, flicking, mixing, and incubating on ice for 30 min; thermally shocking at 42 deg.C for 60s, and standing on ice for 2 min; adding 500 mu L of non-resistant LB liquid culture medium, and performing shake culture at 37 ℃ for 1 h; centrifuging at 4000rpm for 5min, sucking away, reserving about 100 μ L of culture medium, and uniformly blowing the bacterial precipitate by a pipette and then coating the bacterial precipitate on an LB culture medium plate containing ampicillin resistance. The plates were then incubated in an incubator at 37 ℃ for 16 h.

After the clone grows out, selecting a single clone colony to 3mL LB culture solution containing ampicillin, shaking and culturing for 16h at 37 ℃, extracting plasmid DNA, carrying out enzyme digestion identification by Kpn I and Hind III, and obtaining two DNA bands of 4.1kb and 1.2kb in positive clone, as shown in figure 1. The sequence of the positive clone was further confirmed to be completely correct by sequencing.

(2) Constructing salmonella genetic engineering bacteria carrying no-load expression plasmid and salmonella genetic engineering bacteria carrying recombinant expression plasmid with cloned L-methioninase gene.

The no-load expression plasmid pSVSPORT and the recombinant expression plasmid pSVSPORT-L-methioninase are respectively electrically transformed into a VNP20009 strain (YS1646, ATCC No. 202165) to construct salmonella genetically engineered bacteria which are respectively named as VNP20009-V and VNP 20009-M. The specific construction process is as follows:

competent bacteria VNP20009 were placed on ice, and after thawing, they were transferred to a precooled electric rotor, to which 2. mu.L of plasmid was added, gently flicked and mixed, and incubated on ice for 1 min. The electric rotor was placed in an electric rotor under the conditions of 2400V voltage, 400. omega. resistance, 25. mu.F capacitance, and 4ms discharge time. Immediately after the electric shock, 1mL of SOC culture medium was added and mixed gently. Shake culturing at 37 deg.C for 1 h; centrifuging at 4000rpm for 5min, sucking away, reserving about 100 μ L of culture medium, and uniformly blowing the bacterial precipitate by a pipette and then coating the bacterial precipitate on an LB-O culture medium plate containing ampicillin resistance. The plates were then incubated in an incubator at 37 ℃ for 16 h. After VNP20009-V and VNP20009-M are cultured by LB-O, plasmids are extracted, and the restriction enzyme digestion identification is correct.

Take 1X 10⁸Extracting protein from protein lysate of Salmonella, performing 10% SDS-PAGE electrophoresis, transferring to PVDF membrane by ice bath under stable pressure, blocking BSA at room temperature for 1h, rinsing with TBST for 3 × 5min, adding rabbit anti-L-methioninase antibody (1: 1000), and incubating at 4 deg.C overnight. Rinsing with TBST for 3 times, adding HRP-labeled anti-rabbit secondary antibody (1: 10000) for 5min each time, incubating at room temperature for 1h, rinsing with TBST for 3 times, 5min each time, and developing by ECL chemiluminescence method. The results are shown in FIG. 2, and there is a specific band at a molecular weight of about 43kD, indicating that the expression level of L-methioninase is significantly increased in VNP20009-M as compared with VNP20009 and VNP 20009-V.

Mixing L-methionine and pyridoxal with VNP20009, VNP20009-V and VNP20009-M thalli respectively, incubating at 37 ℃ for 10min, stopping with 50% trichloroacetic acid, centrifuging to obtain supernatant, mixing with 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate (MBTH), incubating at 50 ℃ for 30min, measuring absorbance at 320nm, and defining the enzyme amount for catalytically converting 1 mu mol of alpha-ketobutyrate per minute as 1 enzyme activity unit. The results show (FIG. 3) that the methioninase activity of Salmonella VNP20009-M is 10 times higher than that of VNP20009 and VNP 20009-V.

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

1. The mouse L1210 acute T lymphocyte leukemia cells were cultured in DMEM medium containing 10% fetal calf serum, and the number of the cells was 1X 10⁵One was inoculated subcutaneously in the right axilla of nude mice. Every 2 to 3 days, the state of the mice was observed, and the tumor size (volume: 0.5 × length × width) was measured with a vernier caliper²). To be treatedThe tumor volume reaches 80-100 mm³Meanwhile, tumor-bearing nude mice were randomly grouped: PBS group and VNP20009-M group.

2. VNP20009-M was cultured with LB-O, and when OD ≈ 0.6, the cells were collected, resuspended in PBS, and then suspended at 2X 10⁶CFU/dose was administered intratumorally, and control group was injected with PBS of equal volume. After administration, the nude mice were observed for activity, feeding, and body weight. The results are shown in fig. 4, and the body weight of the mice was unaffected after the injection of the bacteria. And the food and the excrement of the nude mice are also not abnormal, which indicates that VNP20009-M has no obvious toxicity to the nude mice.

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

4. Meanwhile, tumor tissues were fixed with 4% formalin overnight, and were paraffin-embedded and sectioned for hematoxylin-eosin (HE) staining. Compared with the PBS control group, pathological section HE staining (figure 9) shows that the tumor after VNP20009-M injection has 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 an anti-tumor effect on HL-60 subcutaneous transplanted tumors.

1. Culturing human HL-60 acute myelogenous leukemia cells with IMDM medium containing 10% fetal calf serum, respectively, at a cell number of 5 × 10⁶One was inoculated subcutaneously in the right axilla of nude mice. Every 2 to 3 days, the state of the mice was observed, and the tumor size (volume: 0.5 × length × width) was measured with a vernier caliper²). When the tumor volume reaches 80-100 mm³Meanwhile, tumor-bearing nude mice were randomly grouped: PBS group and VNP20009-M group.

2. Cultivation of VNP200 with LB-O09-M, when OD ≈ 0.6, the cells were collected, then resuspended in PBS at 2X 10⁶CFU/dose was administered intratumorally, and control group was injected with PBS of equal volume. After administration, 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 unaffected after the injection of the bacteria. And the food and the excrement of the nude mice are also not abnormal, which indicates that VNP20009-M has no obvious toxicity to the nude mice.

3. The length and width of the tumor were measured every 2-3d, the tumor volume was calculated, and a change curve of the tumor volume in nude mice (FIG. 11) and a relative proliferation curve (FIG. 12) were plotted. The results show that the tumor volume of the bacteria-treated group is obviously smaller than that of the control group, and the relative proliferation rate of the tumor is obviously reduced. Mice were sacrificed at the end of treatment, subcutaneous tumors were dissected, photographed (fig. 13), and weighed (fig. 14). The results show that after the salmonella genetically engineered bacterium VNP20009-M is given, the tumor grows slowly, and the volume and the weight are both obviously lower than those of the PBS group. The results all show that VNP20009-M can obviously inhibit the growth of HL-60 transplantable tumor.

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

Example 4: the genetically engineered bacterium VNP20009-M has an antitumor effect on MLL-AF 9-driven AML mice.

1. Taking out MLL-AF9 driven AML cell (with GFP reporter gene) cryopreservation tube from liquid nitrogen tank, rapidly placing in 37 deg.C water bath for resuscitation, washing with sterile PBS for 2 times, performing trypan blue staining, and adjusting cell concentration to 1 × 10⁶After the completion of the reaction, 200. mu.L of the cell lysate, i.e., 1X 10 cells, was collected by mixing with a 1mL syringe⁶Performing mouse tail vein injection on the AML leukemia cells of a C57BL/6J male mouse to induce morbidity;

2. the next day, mice were randomly divided into PBS group and VNP20009-M group; VNP20009-M was cultured with LB-O, and when OD ≈ 0.6, the cells were collected, resuspended in PBS, and then suspended at 2X 10⁶The dose of CFU/water alone is,the drug is administered by tail vein injection, and PBS with the same volume is injected into a control group; after one week, the drug was administered again as above.

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

4. After blood sampling and red blood cell lysis every week, GFP in mouse peripheral blood white cells is detected by flow cytometry⁺Leukemia cell content, i.e. detection of GFP⁺% of the total amount of GFP in the control group (FIG. 17)⁺% was calculated as denominator relative inhibition (fig. 18). The result shows that the VNP20009-M tail vein administration can obviously reduce GFP in mouse peripheral blood leucocyte⁺The leukemia cell content of (a), inhibiting the proliferation thereof, indicates that VNP20009-M has a significant anti-tumor effect on MLL-AF 9-driven AML mice.

The invention shows that the attenuated salmonella typhimurium genetically engineered bacterium VNP20009-M with recombinant expression plasmid capable of cloning and expressing L-methioninase gene can continuously express L-methioninase in tumor tissues, consumes a large amount of methionine and other nutrient substances, causes the tumor cells to lack nutrition and grow slowly, has obvious inhibition effect on the subcutaneous transplanted tumor of acute leukemia cells and MLL-AF9 driven AML, and can be used for preparing the medicine for treating acute leukemia. The above expression vector plasmid is not limited to pSVSPORT plasmid, and pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid and other plasmids having L-methioninase gene cloned therein have similar effects.

Claims

1. The application of the attenuated salmonella typhimurium in preparing the medicine for treating acute leukemia is characterized in that the attenuated salmonella typhimurium is attenuated salmonella typhimurium VNP 20009.

2. An application of a 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 plasmids.

3. The use according to claim 2, wherein the plasmid is a pSVSPORT plasmid, a pTrc99A plasmid, a pcDNA3.1 plasmid, a pBR322 plasmid or a pET23a plasmid.

4. The use according to claim 2 or 3, wherein the plasmid is transferred by electrotransformation into the attenuated Salmonella typhimurium VNP 20009.

5. The use according to claim 4, wherein the electrotransformation conditions are a voltage of 2400V, a resistance of 400 Ω, a capacitance of 25 μ F, and a discharge time of 4 ms.

6. An application of a 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 an L-methioninase gene is cloned on the plasmid.

7. The use of claim 6, wherein the plasmid is pSVSPORT plasmid, pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid.

8. The application of claim 6 or 7, wherein the construction method of the genetically engineered bacteria is as follows: subcloning the L-methioninase gene into a plasmid to obtain an L-methioninase expression plasmid, and electrically transforming the L-methioninase expression plasmid into the attenuated Salmonella typhimurium VNP 20009.

9. The use according to claim 8, wherein the electrotransformation conditions are a voltage of 2400V, a resistance of 400 Ω, a capacitance of 25 μ F, and a discharge time of 4 ms.

10. The use according to claim 1 or 2 or 6, wherein the administration is intratumoral or intravenous.