Attenuated salmonella mutant strain for slyA gene knockout, construction method thereof and application of attenuated salmonella mutant strain in improving melanoma targeting

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: 2017-03-22
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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  • Attenuated salmonella mutant strain for slyA gene knockout, construction method thereof and application of attenuated salmonella mutant strain in improving melanoma targeting
  • Attenuated salmonella mutant strain for slyA gene knockout, construction method thereof and application of attenuated salmonella mutant strain in improving melanoma targeting

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of Attenuated Salmonella slyA Gene Knockout Mutants

[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 pK...

Embodiment 2

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

[0048] The morphological characteristics of △slyA 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 △slyA and VNP20009.

[0049] The attenuated Salmonella mutant strains △slyA 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. Such as figure 1 B, △slyA 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 △slyA was not different from that of VNP20009.

[0050] In the experiment of infecting macrophages in vitro, there was no significant differenc...

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Abstract

The invention discloses an attenuated salmonella mutant strain for slyA gene knockout, and application thereof. The attenuated salmonella mutant strain conducts knockout in attenuated salmonella VNP 20009 on a polynucleotide fragment of which the sequence is SEQ ID N0:2 (including an open reading frame of the slyA gene of which the sequence is SEQ ID N0:1 and six polynucleotide fragments on the upstream of the open reading frame), then a polynucleotide fragment of which the sequence is SEQ ID N0:3 is replaced for the slyA gene. The invention provides the application of the slyA gene deletion attenuated salmonella mutant strain in target mouse melanoma. A high targeting, brand new, safe, low-cost and convenient attenuated salmonella mutant strain of the target mouse melanoma and the 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 slyA 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, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/42
CPCC07K14/255C12N15/74C12N2800/101C12N2800/30
Inventor 华子春张晓昕
Owner NANJING UNIV
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