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Gene deleted attenuated salmonella typhimurium mutant strain and construction method thereof and application of gene deleted attenuated salmonella typhimurium mutant strain in improvement of melanoma targeting performance

A Salmonella and construction method technology, applied in the field of microorganisms, can solve the problems of ineffective targeting of tumor tissue, etc., and achieve the effect of good tumor treatment application prospects, high tumor targeting, and low cost

Active Publication Date: 2017-02-22
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although VNP20009-mediated gene therapy has achieved certain therapeutic effects, and its safety has been verified in phase I clinical trials, the results of clinical trials have shown that VNP20009 cannot effectively target tumor tissue and inhibit tumor growth, so it is necessary to Optimizing the VNP20009 Bacterial Therapeutic Vector

Method used

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  • Gene deleted attenuated salmonella typhimurium mutant strain and construction method thereof and application of gene deleted attenuated salmonella typhimurium mutant strain in improvement of melanoma targeting performance
  • Gene deleted attenuated salmonella typhimurium mutant strain and construction method thereof and application of gene deleted attenuated salmonella typhimurium mutant strain in improvement of melanoma targeting performance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of attenuated Salmonella STM3120 gene knockout mutant

[0034] (1) Preparation of attenuated Salmonella VNP20009 competent cells by electroporation

[0035]Attenuated Salmonella was inoculated in LB medium, and cultured with vigorous shaking on a shaker at 37°C until OD600=0.5; quickly cooled the bacterial solution on ice for 10 minutes, and refrigerated and centrifuged at 5000 rpm / min at 4°C for 4 minutes; discarded the supernatant , add 1ml of ice-cold 10% glycerol, mix gently with a pipette gun to resuspend the cells, and centrifuge at 5000rpm / min at 4°C for 4 minutes; discard the supernatant, add 1ml of ice-cold 10% glycerol, and use a pipette gun Gently mix to resuspend the cells, refrigerate and centrifuge at 5000rpm / min at 4°C for 4 minutes; add 80 μl ice-cold 10% glycerol, mix gently with a pipette to resuspend the cells, use immediately or place quickly in- Store at ultra-low temperature at 70°C.

[0036] (2) Electric transformation of ...

Embodiment 2

[0047] Example 2 Comparison of Biological Properties of Attenuated Salmonella Mutant Strain △STM3120 and VNP20009

[0048] The morphological characteristics of △STM3120 and VNP20009 were observed by scanning electron microscope. Statistical analysis of the length and width of bacteria by electron microscope pictures ( figure 1 A), There is no significant difference in the morphological characteristics of △STM3120 and VNP20009.

[0049] The attenuated Salmonella mutant strains △STM3120 and VNP20009 were respectively cultured in LB medium, the growth curves were drawn, and the growth curves of the two in the medium were compared. like figure 1 B, △STM3120 and VNP20009 both showed exponential growth under the optimal culture conditions in vitro, and the growth curves of the two basically overlapped, which indicated that the in vitro growth rate of △STM3120 was not different from that of VNP20009.

[0050] In the experiment of infecting macrophages in vitro, the infection rate ...

Embodiment 3

[0057] Example 3 Attenuated Salmonella Mutant Strains Analysis of Bacterial Titers in Tumor and Spleen of Melanoma-bearing Mice

[0058] In order to determine the biological distribution of attenuated Salmonella △STM3120 and VNP20009 in tumor-bearing mice, when the tumor volume of tumor-bearing mice reached 150 mm 3 Each mouse was intraperitoneally injected with 5×10 5 CFU of attenuated Salmonella, 3 days after bacterial infection. The tumors and spleens of the mice were removed under aseptic conditions and analyzed for the number of attenuated Salmonella. The results are shown in the figure: in the tumor tissue, the number of colonies of the attenuated Salmonella △STM3120 and VNP20009 strains was similar, and the results were not statistically different, which indicated that they had similar tumor targeting capabilities ( figure 2 A). At the same time, the number of △STM3120 in spleen was significantly lower than that of VNP20009 strain (P figure 2 B). In addition, bacte...

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Abstract

The invention discloses an attenuated salmonella typhimurium mutant strain with STM3120 genes knocked out and application thereof. The attenuated salmonella typhimurium mutant strain is a polynucleotide fragment with attenuated salmonella typhimurium VNP20009 knocked out, the sequence of the polynucleotide fragment is SEQ ID NO:2, and the polynucleotide fragment is replaced with a polynucleotide fragment with the sequence of SEQ ID NO:3. Application of the attenuated salmonella typhimurium mutant strain with the STM3120 genes knocked out in targeted mouse melanoma is disclosed. The mouse melanoma targeted attenuated salmonella typhimurium mutant strain which is high in targeting performance, brand-new, safe, low in price and convenient to use and application thereof are provided.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to the construction of an attenuated Salmonella mutant strain with STM3120 gene deletion and its application in targeting mouse melanoma. Background technique [0002] Malignant tumors are a type of disease that seriously threatens human life and health. According to the report of the World Health Organization, there were about 14 million new cancer cases and 8.2 million cancer-related deaths in 2012, and it is estimated that the number of new cases will increase by about 70% in the next two decades. %. The current main methods of tumor treatment include surgery, chemotherapy and radiotherapy, but they all have serious deficiencies in the treatment of tumors. The scope of application of surgery is narrow. If the tumor tissue exceeds local or regional lymph nodes, direct resection cannot be cured well; chemotherapy and radiotherapy have low targeting, insufficient tissue penetration, a...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12R1/42
CPCC12N15/74C07K14/255C12N2800/101C12N2800/30
Inventor 华子春张晓昕
Owner NANJING UNIV
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