APPLICATION OF GENETICALLY GENINEERED BACTERIUM OF ATTENUATED SALMONELLA TYPHIMURIUM IN PREPARATION OF MEDICINE FOR TREATMENT OF LIVER CANCER

Abstract

Provided is an application of a genetically engineered bacterium of attenuated Salmonella typhimurium in preparation of medicines for treating liver cancer. The bacterium is attenuated Salmonella typhimurium VNP20009 carrying a plasmid cloned with a methioninase gene. Also provided is a construction method of the bacterium.

Claims

1. A method for treating liver cancer, the method comprising administering a genetically engineered bacterium to a human having liver cancer, wherein the genetically engineered bacterium is attenuated Salmonella typhimurium VNP20009 carrying a plasmid, and the plasmid is cloned with a L-methioninase gene.

2. The method 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 method according to claim 1, wherein the genetically engineered bacterium is constructed by: subcloning the L-methioninase gene into the plasmid, then electro-transforming the plasmid to attenuated Salmonella typhimurium VNP20009.

4. The method according to claim 3, wherein the electrotransformation condition is as follows: voltage 2400V, resistance 400 , capacitance 25 F, discharge time 4 ms.

5. The method according to claim 3, wherein the plasmid is pSVSPORT plasmid and the L-methioninase gene is cloned to the plasmid by Kpn I and Hind III restriction sites, then electro-transforming the plasmid to attenuated Salmonella typhimurium VNP20009.

6. The method according to claim 1, wherein the the genetically engineered bacterium is administered via intravenous or interventional injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows the 1% agarose gel electrophoresis of plasmid pSVSPORT-L-methioninase digestion assay.

[0015] FIG. 2 shows the Western blot analysis of methioninase expression.

[0016] FIG. 3 shows the detection of methioninase activity in salmonella.

[0017] FIG. 4 shows the curve of tumor volume change after administration of Salmonella.

[0018] FIG. 5 shows the tumor size of mice anesthetized 4 weeks after administration of Salmonella. The black frame indicates the tumor.

[0019] FIG. 6 shows the tumor size 4 weeks after administration of Salmonella.

[0020] FIG. 7 shows the tumor weight 4 weeks after administration of Salmonella.

[0021] FIG. 8 shows the curve of tumor volume change after administration of L-methioninase.

DETAILED DESCRIPTION

[0022] The present invention can be better understood from the following examples. However, it will be readily understood by those skilled in the art that the embodiments described are intended to be illustrative of the invention, not and should not be construed as limiting the invention as set forth in the claims.

Example 1: Construction of Genetic Engineering Bacterium

[0023] (1) Construction of a Plasmid Expressing the L-Methioninase Gene

[0024] The L-methioninase (GenBank: L43133.1) gene is synthesized and subcloned to pUC57 plasmid (Genscript), then subcloned to pSVSPORT plasmid (invitrogen) through the Kpn I and Hind III restriction sites, to get the pSVSPORT-L-methioninase expression plasmid. The specific procedure is as follows:

[0025] The pSVSPORT plasmid was digested with Kpn I and Hind III, with the digestion system: 2 g of plasmid DNA, 3 mL of 10 buffer, 1.5 L of Kpn I enzyme, 1.5 L of Hind III enzyme, added with ddH2O to 30 L, incubate warm bath for 3 h at 37 C., then the digestsion system was separated by 1% agarose gel electrophoresis in 1% agarose gel, to cut out DNA bands at awith the size of 4.1 kb, then DNA was purified by gel recovery and purification kit.

[0026] DNA fragments of L-methioninase coding region were obtained by gene synthesis and subcloned to pUC57 plasmid (Genscript), digested with Kpn I and Hind III, with the digestion system: 3 g of plasmid DNA, 3 mL of 10 buffer, 1.5 L of Kpn I enzyme, 1.5 L of Hind III enzyme, added with ddH2O to 30 L, warm bath for 3 h at 37 C., then the digestion system was separated by 1% agarose gel electrophoresis in 1% agarose gel, to cut out DNA bands at awith the size of 1.2 kb, then DNA was purified by gel recovery and purification kit.

[0027] The pSVSPORT (Kpn I/Hind III) and DNA fragment of the L-methioninase coding region (Kpn I/Hind III) were ligated. The ligation reaction condition: 2 mL of vector, 6 L of inserted fragment, 1 L of T4 DNA ligase, water bath for 16 h at 16 C.

[0028] The ligation product was transformed into competent cells of E. coli DH5a (Takara). One tube of 50 mL DH5a competent cells was placed on the ice until thawing molten, then 5 L of above ligation product was added, mixed well by flicking, incubated on ice for 30 min; after heat shock 60 s at 42 C., placed on ice for 2 min; then 500 L of non-resistant LB liquid medium was added and incubated at 37 C. for 1 h with shaking, then spread on ampicillin resistant LB medium plate and cultured overnight.

[0029] When clones grew, single clone was innoculated to 3 mL of ampicillin-containing LB medium, incubated at 37 C. for 16 h. The plasmid DNA was extracted and identified by Kpn I and Hind III digestion. Two DNA bands at the size of 4.1 kb, 1.2 kb were obtained in the positive clones, as shown in FIG. 1. The sequence of the positive clones was further confirmed by sequencing.

[0030] (2) Construction of Plasmid-Bearing VNP20009 Strain and VNP20009 Strain Bearing a Plasmid Cloned with L-Methioninase Gene

[0031] The pSVSPORT and pSVSPORT-L-methioninase expression plasmids are electro-transformed to VNP20009 strain (YS1646, ATCC No. 202165) respectively, and named as VNP20009-V and VNP20009-M, respectively. The specific construction process is as follows:

[0032] The competent bacteria VNP20009 was placed on ice, after melted, transferred to a pre-cooled electric rotating-cup and 2 mL of the plasmid was added, mixed well by flicking, incubated on ice for 30 min; after heat shock 60 s at 42 C., placed on ice for 1 min. The electric rotating-cup was placed into an electroporator, and the condition was set to voltage 2400V resistance 400, capacitance 25 F, discharge time 4 ms. After the electric shock, 1 mL SOC medium was added and mixed well gently, incubated at 37 C. for 1 h with shaking. After the bacterial precipitation was blown by a pipette and uniformly spread on an ampicillin-resistant LB-O medium plate, then incubated 16 h at 37 C. incubator. After the VNP20009-V and VNP20009-M were cultured with LB-O, the plasmids were extracted and identified by restriction enzyme digestion.

[0033] The protein was extracted from 110.sup.8 Salmonella and separate by 10% SDS-PAGE electrophoresis, transferred to PVDF membrane under constant voltage, after blocked 1 h with BSA at room temperature, rinsed 35 min with TBST, added with the rabbit anti-L-methioninase antibody (1:1000) overnight at 4 C., rinsed with TBST 3 times, 5 min each time, then HRP labeled anti-rabbit secondary antibody (1:10000) was added, incubated at room temperature for 1 h, rinsed with TBST 3 times, 5 min each time, developed using the enhanced chemiluminescent (ECL). The results are shown in FIG. 2. Specific bands were found at about 43 kD molecular weight, indicating that the expression of L-methioninase significantly increased in VNP20009-M compared with that in VNP20009 and VNP20009-V.

[0034] L-methionine and pyridoxal were mixed with VNP20009-V and VNP20009-M strains respectively, and incubated at 37 C. for 10 min. After terminated by 50% trichloroacetic acid, the mixed solution was centrifuged to get the supernatant, then well mixed with 3-methyl-2 MBTH; after incubated at 50 C. for 30 min, the absorbance at 320 nm was determined. The amount of enzyme that catalyzes to covert -ketobutyric acid was defined as one unit of enzyme activity. The results are shown in FIG. 3. The methioninase activity of Salmonella VNP20009-M was 10 times higher than that of VNP20009-V.

Example 2: Anti-Tumor Effect of VNP20009-L-Methioninase Strain

[0035] 1. Highly metastatic HCC cell HCCLM3 is cultured in DMEM medium containing 10% fetal bovine serum. 210.sup.6 cells are inoculated subcutaneously on the right armpit of nude mice. The tumor-bearing nude mice are randomized as: PBS control group, VNP20009-V group and VNP20009-M group.

[0036] 2. VNP20009-V and VNP20009-M are cultured with LB-O. When OD0.6, the cells are harvested and resuspended in PBS. On the third day after inoculation, mice are administered at a dose of 110.sup.4 CFU/g (about 210.sup.5 CFU/mouse) by tail vein injection while the control group are administered with the same volume of PBS. The mice are observed every 2-3 days after administration. The tumor size is measured by a vernier caliper (volume=0.52lengthwidth.sup.2), and the tumor volume change curves of nude mice are ploted (FIG. 4). On the 30th day after administration, three mice are randomly selected from each group to anesthetize and photograph (FIG. 5). Two mice are randomly selected from the control group and test group separately. The tumors of nude mice are dissected, weighed and photographed (FIGS. 6, 7). The results are shown in FIGS. 4, 5. After modeling, the tumors of the mice in the PBS and blank groups grow normally and increase quickly; while after administration of Salmonella VNP20009-M, the tumors are shrank or even completely disappear in some mice. The growth of tumors stops in most mice in the VNP20009-M group, and the tumor volume and weight (FIGS. 6, 7) are about of those in the VNP20009-V group and of those in the PBS group. These results show that Salmonella VNP20009-M has a significant inhibitory effect on the liver tumor.

[0037] 3. The procedures are the same as those in 1. Tumor-bearing nude mice are divided into two groups and administered with PBS or L-methioninase at a dose of 100 ng/mouse respectively by intravenous injection. The tumor size is measured by a vernier caliper (volume=0.52lengthwidth.sup.2) and the tumor volume change curves of nude mice are ploted. As shown in FIG. 8, there is no significant difference in the tumor size between two groups. The dose of L-methioninase at 1 ng/mouse is equivalent to that of L-methioninase contained in 210.sup.6 CFU VNP20009-M. Thus, a 100-fold dose of L-methioninase has no significant anti-tumor effect. This indicates that, with the depletion or degradation of L-methioninase, a single administration does not function, while the sustained high expression of L-methioninase using VNP20009 as a carrier can make up this defect, showing a significant anti-tumor effect.

[0038] The invention has showed that genetic engineering bacterium has a significant inhibitory effect on HCC cells. The attenuated Salmonella typhimurium VNP20009 carrying a plasmid cloned with a L-methioninase gene can continuously express L-methioninase in the liver tumor tissues, which consumes a large amount of methionine and other nutrients, so that the tumor cells are lack of nutrition and grow slowly. Therefore, it can be used in the manufacture of medicaments for treating liver cancer. The plasmid is not limited to a pSVSPORT plasmid. The pTrc99A plasmid, pcDNA3.1 plasmid, pBR322 plasmid or pET23a plasmid and the above plasmids cloned with L-methioninase gene have similar effects.