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Salmonella strain used for RNA interference in mammalian cells, preparation method and application of salmonella strain

A technology of RNA interference and Salmonella, which is applied in the field of microorganisms, can solve the problems of not being able to serve as an RNA interference delivery system, reduce the survival rate of bacteria, and reduce the survival ability, and achieve excellent safety, difficulty in survival, and increased bacterial titer.

Active Publication Date: 2015-11-25
JIANGSU TARGET BIOMEDICINE RES INST +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. The phoP / phoQ gene knockout bacteria are no longer wrapped by intracellular vesicles in macrophages, so the survival rate of the bacteria is significantly reduced, which directly limits the application of phoP / phoQ gene knockout bacteria
[0007] 2. Bacteria knocked out of phoP / phoQ gene are less survivable in cells such as macrophages, which leads to a significant reduction in the toxicity of bacteria to BALB / c mice and other animals, but they still have certain toxic and side effects on animals
[0009] 4. Although the attenuated Salmonella VNP20009 can be used as a delivery vehicle for gene expression vectors, it cannot be used as a delivery system for RNA interference
[0010] 5. Although the attenuated Salmonella VNP20009 has a high targeting ability to hypoxic tissues (such as tumors, etc.) when applied in vivo, it still has a certain distribution in normal tissues such as spleen and liver, which leads to potential side effects (ChenG et al., CancerScience 2009; 100:2437-2443)

Method used

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  • Salmonella strain used for RNA interference in mammalian cells, preparation method and application of salmonella strain
  • Salmonella strain used for RNA interference in mammalian cells, preparation method and application of salmonella strain
  • Salmonella strain used for RNA interference in mammalian cells, preparation method and application of salmonella strain

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0053] VPN (purI - / msbB - / phoP - / phoQ - ) construction

[0054] (1) Preparation of recombinant DNA fragments

[0055] Taking the preparation method constructed on the basis of the VNP20009 strain as an example, the construction of VNP (phoP / Q-) is carried out according to the classic method reported in the literature (DatsenkoKA et al., ProcNatlAcadSciUSA2000; 97: 6640-6645), as follows: The phoP / phoQ gene on the Salmonella chromosome was replaced by a gene fragment amplified by PCR with kanamycin (Kna) resistance. This kanamycin-resistant fragment was amplified by PCR using the pkd4 plasmid as a template, and the primer sequences used were as follows:

[0056] PhoP-F:

[0057] 5'-CACCATAATCAACGCTAGACTGTTCTTATTGTTAACACAAGGGAGAAGAGGTGTAGGCTGGAGCTGCTTC-3',

[0058] phoQ-R:

[0059] 5'-CGATTATAACGGATGCTTAACGAGATGCGTGGAAGAACGCACAGAAATGTCATATGAATATCCTCCTTAG-3'.

[0060] The 5' end of the above primers contains 40-50 base pairs homologous to the phoP / phoQ gene. The PCR pr...

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Abstract

The invention discloses a salmonella strain used for RNA interference in mammalian cells. The salmonella strain is a salmonella mutant strain containing purI, msbB, phoP and phoQ gene knockout. In addition, the invention further discloses a preparation method and application of the salmonella strain.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and relates to a Salmonella strain used for RNA interference in mammalian cells, a preparation method and application thereof. Background technique [0002] RNA interference technology has become an effective research tool for silencing specific genes, and RNA interference technology also has great application prospects in the treatment of cancer and other diseases (LiebermanJ et al., Trends MolMed 2003; 9:397-403). RNA interference technology can target and degrade a specific RNA to regulate cell proliferation and apoptosis (LakkaSS et al., Oncogene2004; 23:4681-4689; GondiCS et al., CancerRes2004; 64:4069-4077; FlemingJB et al., MolCancerRes2005; 3:413-423). However, how to transport RNA interference fragments to the cytoplasm of cells hinders the development and application of RNA interference technology. Although viral vectors (DevroeE et al., ExpertOpinBiolTher2004; 4:319-327; Kargi...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/09A61K35/74A61K48/00A61P35/00C12R1/42
Inventor 华子春程侠为张晓昕
Owner JIANGSU TARGET BIOMEDICINE RES INST
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