A genetically engineered bacterium that inhibits tumor growth and its construction method and application

A technology of genetically engineered bacteria and its construction method, which is applied in the field of genetically engineered bacteria that inhibit tumor growth and its construction, can solve the problems of insignificant anti-tumor effect and poor enrichment ability of anti-tumor genetically engineered bacteria, and achieve good anti-tumor, Strong tumor targeting and high safety effect

Active Publication Date: 2022-08-02
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of poor enrichment ability and insignificant anti-tumor effect of existing anti-tumor genetically engineered bacteria, the present invention provides a genetically engineered bacteria that inhibits tumor growth and its construction method and application, by linking the DNA sequence encoding Sox2shRNA to Construct a recombinant plasmid in the vector plasmid pGPU6, and then transform the recombinant plasmid into attenuated Salmonella typhimurium VNP20009 to obtain genetically engineered bacteria

Method used

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  • A genetically engineered bacterium that inhibits tumor growth and its construction method and application
  • A genetically engineered bacterium that inhibits tumor growth and its construction method and application
  • A genetically engineered bacterium that inhibits tumor growth and its construction method and application

Examples

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Effect test

Embodiment 1

[0036] Construction of genetically engineered bacteria

[0037] (1) Construction of shRNA recombinant expression plasmid pGPU6-shSox2 targeting gene Sox2

[0038] Firstly, shRNA targeting Sox2 (shSox2, SEQ ID NO: 1) was designed according to the sequence of Sox2 gene (Gene ID: 6657), and the upstream of the shSox2 sequence (SEQ ID NO: 2) was synthesized in Gene Synthesis Company (Shanghai Gema Pharmaceutical Technology Co., Ltd.). ) and downstream (SEQ ID NO: 3) primer DNA fragments. At the same time, as the negative control group of the experiment, a control shRNA (shCon, SEQ ID NO: 4) expression sequence that cannot target any gene was designed, and the upper (SEQ ID NO: 5) and downstream (SEQ ID NO: 6) primer DNA fragments were synthesized. . According to the OD value of the synthesized DNA fragments, add deionized water to dilute the DNA concentration to 20 μM. The annealing reaction process is: after mixing 5 μL of upstream primer and 5 μL of downstream primer, add 5 μ...

Embodiment 2

[0047] MTT assay detects the inhibitory effect of the genetically engineered bacteria of the present invention on the growth of various human tumor cells

[0048] The MTT method was used to detect the inhibitory effect of the genetically engineered bacteria of the present invention on the growth of various human tumor cells. at 37°C, 5% CO 2 After culturing the tumor cells to a density of 90% in an incubator, they were harvested by 0.25% trypsinization, and the cells were resuspended in cell culture medium and counted, and the cell concentration was adjusted to 2.0 × 10 4 cells / mL, inoculate the cell suspension into a 96-well plate, 100 μL per well, and inoculate at 37°C, 5% CO 2 Incubate overnight in an incubator. After the cells adhered, the genetically engineered bacteria of the present invention (1.0×10 6 cfu), the genetically engineered bacteria of the present invention and the vascular inhibitor bevacizumab (2.5 μg) combined administration group, the genetically engin...

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Abstract

The invention discloses a genetically engineered bacterium for inhibiting tumor growth, a construction method and application thereof, and belongs to the field of biotechnology. The recombinant plasmid was transformed into attenuated Salmonella typhimurium VNP20009. The preparation method of the genetically engineered bacteria is simple and easy to operate. The invention also provides the construction method of the genetically engineered bacteria and the application of the bacteria in the drug for the treatment of tumors. When the genetically engineered bacteria of the present invention is combined with vascular inhibitor drugs to treat tumors at the same time, the combined administration has a significant anti-tumor effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more particularly, relates to a genetically engineered bacterium for inhibiting tumor growth and a construction method and application thereof. Background technique [0002] Cancer stem cells are the root cause of tumor growth, drug resistance, and tumor recurrence. The characteristics of cancer stem cells are: the ability to continue to proliferate, the ability of a small number of cells to form tumors in vivo, the ability to resist drugs, the ability to be cultured in vitro without serum into spheres (sphere formation), and the ability to differentiate. Many proteins that play important functions in embryonic stem cells are also highly expressed and activated in cancer stem cells. Sox2 (sex-determining region Y-box2) is a key gene for maintaining stem cell diversity, and it is highly expressed in a variety of tumor cells and drug-resistant tumor cells. shRNA (shorthairpin RNA) interference te...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74A61K48/00A61K31/7088A61P35/00C12R1/42A61K39/395A61K31/404
CPCA61K39/39558A61K31/404A61K31/7088C12N15/113C12N15/74C07K14/47C12N2310/531C12N2310/14C12N2800/101A61K2300/00
Inventor 徐寒梅赵长宏
Owner CHINA PHARM UNIV
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