siRNA-DNA nanometer system for targetedly inhibiting Salmonella drug-resistance efflux pump gene acrA, and preparation method thereof

A nano and nano-carrier technology, applied in the field of siRNA-DNA nano system, can solve the problems of short effective time, expensive reagents, long experiment period, etc.

Active Publication Date: 2018-03-16
SICHUAN UNIV
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Problems solved by technology

Since naked siRNA is easily degraded, unstable, and has a short effective action time, in order to prolong the action time of siRNA, most of the siRNA silencing bacterial genes are delivered by expression vectors or transfection reagents, but this method has a long experimental period and expensive reagents, which is not feasible. Targeting, requires high concentration of excess siRNA, and easily leads to off-target

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  • siRNA-DNA nanometer system for targetedly inhibiting Salmonella drug-resistance efflux pump gene acrA, and preparation method thereof
  • siRNA-DNA nanometer system for targetedly inhibiting Salmonella drug-resistance efflux pump gene acrA, and preparation method thereof
  • siRNA-DNA nanometer system for targetedly inhibiting Salmonella drug-resistance efflux pump gene acrA, and preparation method thereof

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[0043] 1. Materials and instruments

[0044] 1.1 Strains

[0045] Chloramphenicol-susceptible Salmonella S1 isolates, chloramphenicol-resistant Salmonella typhimurium R5 isolates with a multidrug-resistant phenotype and positive for RND efflux pump family resistance genes, and chloramphenicol-resistant but negative for associated resistance genes , Salmonella Enteritidis R2 isolates and quality control strain Escherichia coli (E.coli, ATCC 25922) were all preserved by the Sichuan Provincial Key Laboratory of Animal Disease Control and Food Safety, Sichuan University.

[0046] 1.2 Main reagents

[0047] Trizol kit was purchased from Invitrogen;

[0048] Taq enzyme, T4DNA ligase, dNTP, reverse transcription kit RevertAid TM First strand cDNASynthesis Kit was purchased from Fermentas;

[0049] All the DNA sequences designed and used were synthesized by Shanghai Sangon Biotechnology Co., Ltd. by conventional methods in the industry;

[0050] 50×TAE / Mg 2+ Buffer: Weigh 242.2...

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Abstract

The invention belongs to the technical field of research on the drug resistance of microorganisms, and provides a siRNA-DNA nanometer system for targetedly inhibiting Salmonella drug-resistance effluxpump gene acrA, and a preparation method thereof, specifically a DNA nanometer vector, which has a tetrahedron-like structure formed by assembling an oligonucleotide A single chain, an oligonucleotide B single chain, and an oligonucleotide C single chain. Based on the DNA nanometer vector, the present invention further provides a nanometer system, a preparation method and applications thereof, wherein the DNA nanometer vector loads a siRNA targeting efflux pump gene to from the nanometer system. According to the present invention, the DNA nanometer vector has the tetrahedron-like structure, can stably load siRNA, and can safely, rapidly and efficiently transport the siRNA to target cells so as to inhibit the expression of the Salmonella efflux pump gene, such that the sensitivity on antibiotics by Salmonella is improved so as to effectively inhibit the drug resistance of Salmonella.

Description

technical field [0001] The invention belongs to the technical field of microbial drug resistance research, and in particular relates to a DNA nanocarrier, an siRNA-DNA nanosystem for targeting and inhibiting Salmonella drug resistance efflux pump gene acrA, a preparation method and application thereof. Background technique [0002] Salmonella is a very important class of Gram-negative bacteria that can cause zoonotic diseases. The problem of Gram-negative bacteria, especially Salmonella antibiotic resistance is increasing, which has become one of the hot spots of global concern. The failure of antibiotic treatment of bacterial infections is increasing due to the continuous spread of multi-drug resistant Gram-negative bacteria. The World Health Organization (WHO) declares that drug-resistant bacteria have become one of the main factors threatening human health, making it more urgent and meaningful to find ways to solve bacterial resistance. Studies have shown that efflux pu...

Claims

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Application Information

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IPC IPC(8): C12N15/87C12N1/21C12N15/113C12R1/42
CPCC07K14/255C12N15/113C12N15/87C12N2310/141
Inventor 王红宁张安云张鹏雷昌伟
Owner SICHUAN UNIV
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