Application of attenuated salmonella typhimurium genetic engineering bacterium in preparation of drug for treating liver cancer

A genetically engineered bacterium, a technology for treating liver cancer, applied in the field of medicine, can solve problems such as side effects and immune reactions, achieve strong anti-tumor activity, simple preparation method, and good application prospects

Active Publication Date: 2017-03-08
GUANGZHOU SINOGEN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since mammals themselves do not express methionase, the way of exogenous administration has certain side effects, often causing the body's immune response

Method used

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  • Application of attenuated salmonella typhimurium genetic engineering bacterium in preparation of drug for treating liver cancer
  • Application of attenuated salmonella typhimurium genetic engineering bacterium in preparation of drug for treating liver cancer
  • Application of attenuated salmonella typhimurium genetic engineering bacterium in preparation of drug for treating liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: the construction of genetically engineered bacteria.

[0023] (1) Construction of a plasmid expressing the L-methioninase gene.

[0024] Synthesized L-methioninase (GenBank: L43133.1) gene was subcloned into pUC57 plasmid (GenScript), and then subcloned into pSVSPORT plasmid (invitrogen) through Kpn I and Hind III restriction sites to obtain pSVSPORT-L- methioninase expression plasmid. The specific construction process is as follows:

[0025] Digest the pSVSPORT plasmid with Kpn I and Hind III, the enzyme digestion system is: 2 μg plasmid DNA, 3 μL 10× buffer, 1.5 μL Kpn I enzyme, 1.5 μL Hind III enzyme, add ddH 2 Make up the volume with O to 30 μL, and incubate at 37°C for 3h. Then the digestion system was separated by electrophoresis in 1% agarose gel, the DNA band with a size of 4.1 kb was cut out, and the DNA was purified with a gel recovery and purification kit.

[0026] The DNA fragment of the L-methioninase coding region obtained by whole gene s...

Embodiment 2

[0035] Example 2: Antitumor effect of VNP20009-L-methioninase strain.

[0036] 1. Cultivate highly metastatic liver cancer cells HCCLM3 with DMEM medium containing 10% fetal bovine serum, with a cell number of 2×10 6 The tumor-bearing nude mice were subcutaneously inoculated into the right axilla of nude mice, and randomly divided into PBS control group, VNP20009-V group and VNP20009-M group.

[0037] 2. Cultivate VNP20009-V and VNP20009-M with LB-O, when OD≈0.6, collect the bacteria, and then resuspend with PBS. On the 3rd day after tumor seeding, 1×10 4 CFU / g (about 2×10 5 CFU / rat) were administered by tail vein injection, and the control group was injected with the same volume of PBS. Every 2 to 3 days after administration, the state of the mice was observed, and the tumor size was measured with a vernier caliper (volume = 0.52 × length × width 2 ), draw the curve of tumor volume change in nude mice ( Figure 4 ). On the 30th day after administration, 3 mice were sele...

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Abstract

The invention discloses an application of an attenuated salmonella typhimurium genetic engineering bacterium in preparation of a drug for treating liver cancer; the bacterium is attenuated salmonella typhimurium VNP20009 cloned with an L-methioninase gene; the attenuated salmonella typhimurium with a plasmid cloned with the L-methioninase gene can continuously express L-methioninase in a liver cancer tumor tissue, a lot of methionine and other nutrient substances are consumed, and tumor cells are lack of nutrition and grow slow. The invention also discloses a construction method and an application of the genetic engineering bacterium. A biological drug for treating liver cancer is a novel safe and toxic-free biological drug having antitumor activity, takes the attenuated salmonella typhimurium VNP20009 as a vector, highly expresses the methioninase by a gene recombination technology, has strong anti-liver cancer tumor activity, and can meet the use demand. The preparation method is simple and easy to operate, and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of medicine, in particular to the application of genetically engineered bacteria of attenuated Salmonella typhimurium in the preparation of drugs for treating liver cancer. Background technique [0002] Liver cancer is one of the most common malignant tumors in the world. China is the country with the highest incidence rate of liver cancer in the world, ranking second only to lung cancer among common tumors. The incidence of liver cancer in China accounts for 45% of the world's total, and the mortality rate ranks third among malignant tumors. Treatment for hepatocellular carcinoma includes surgery, radiation therapy, and chemotherapy. Due to the poor sensitivity of liver cancer to radiotherapy, and the treatment of conventional chemotherapy drugs (such as doxorubicin, fluorouracil, cisplatin) not only has serious side effects, but also cannot significantly alleviate the disease, so the current treatment of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P35/00C12N1/21C12N15/74
CPCC12N9/88C12Y404/01011C12N15/00A61P1/16A61P35/00A61K9/0019C12N15/74A61K35/74C12N15/63A61K39/02C12N1/20
Inventor 赵子建周素瑾林艳赵正刚李芳红
Owner GUANGZHOU SINOGEN PHARMA CO LTD
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