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siRNA for inhibition of microorganism drug resistance and application of same

A microbial, drug-resistant technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as gene-specific silencing

Pending Publication Date: 2017-04-26
GUANGDONG UNIV OF PETROCHEMICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNAi is a very effective method to reduce gene expression, but in the early days, the application of RNAi was limited to plants and animals C. elegans and Drosophila, which is due to the effective induction of gene-specific silencing by long-chain dsRNA in these organisms

Method used

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  • siRNA for inhibition of microorganism drug resistance and application of same
  • siRNA for inhibition of microorganism drug resistance and application of same
  • siRNA for inhibition of microorganism drug resistance and application of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, siRNA

[0031] Through a large amount of experimental research and screening in the early stage, the present invention finally found that 3 kinds of siRNA have good effects on the capture of the drug-resistant gene cassette and the expression of the drug-resistant gene by the integrase gene in the silencing and interference bacterial integron system (high efficiency and specificity) ), the specific sequences of these three siRNAs are as follows.

[0032] siRNA-1:

[0033] siRNA-1 sense strand: 5'-GCACAUGCGUGUAAAUCAUTT-3' (SEQ ID NO: 1),

[0034] siRNA-1 antisense strand: 5'-AUGAUUUACACGCAUGUGCTT-3' (SEQ ID NO: 2),

[0035] siRNA-2:

[0036] siRNA-2 sense strand: 5'-CCUGUUCGGUUCGUAAACUTT-3' (SEQ ID NO: 3),

[0037] siRNA-2 antisense strand: 5'-AGUUUACGAACCGAACAGGTT-3' (SEQ ID NO: 4),

[0038] siRNA-3:

[0039] siRNA-3 sense strand: 5'-CACGGUUCGAAUGUCGUAATT-3' (SEQ ID NO: 5),

[0040] siRNA-3 antisense strand: 5'-UUACGACAUUCGAACCGUGTT-3' (SEQ ID NO: 6)....

Embodiment 2

[0042] Example 2 Construction of siRNA interference vector

[0043] 1) siRNA annealing

[0044] The synthesized siRNA sense strand and antisense strand of Example 1 were dissolved in TE to a solution with a concentration of 100 μM, and an annealing reaction system was prepared according to the following table.

[0045]

[0046] Place the above annealing reaction system on a PCR instrument for annealing treatment: 95°C, 5min; 85°C, 5min; 75°C, 5min; 70°C, 5min; store at 4°C to obtain shRNA fragments.

[0047] 2) Construction of pGPU6 / GFP / Neo-siRNA expression vector

[0048] The vector map of pGPU6 / GFP / Neo (purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.) is as follows figure 1 As shown, the pGPU6 / GFP / Neo vector was digested with Bbs Ⅰ and BamH Ⅰ to obtain the target linear fragment, and then the shRNA fragment obtained above was ligated with the digested pGPU6 / GFP / Neo. The connection system is as follows:

[0049]

[0050] After reacting at 22°C for 1 ...

Embodiment 3

[0051] Example 3 Detection of siRNA interference efficiency

[0052] experimental method:

[0053] In order to detect the interference efficiency of the siRNA of the present invention, the siRNA-1, siRNA-2, and siRNA-3 interference vectors prepared in Example 2 were transfected to cause drug-resistant bacteria Escherichia coli E.coli BL21 (DE3). The integrase contained in Escherichia coli is IntI1, and the drug resistance gene cassette contained in it is aadA1. The transfection method is as follows: adding siRNA three times, adding once every 2 hours, the final concentration of the co-cultivation system is 0.2 μM siRNA, and the co-cultivation time is 8 hours. RT-PCR quantitatively detected the mRNA expression levels of the target genes (integrase IntI1, drug-resistant gene box aadA1) in the drug-resistant Escherichia coli E. coli BL21 (DE3) before and after siRNA treatment; control group.

[0054] The above RT-PCR system and program are:

[0055] Use Bio-Bad CFX 96 Real-ti...

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Abstract

The invention discloses siRNA for inhibition of microorganism drug resistance and an application of same. The siRNA, when being introduced into a drug-resistance bacterial strain, can silence and interfere in capture of a drug-resistance gene cassette by an integrase gene in an integron system and expression of a drug-resistance gene. Through RT-PCR quantitative detection on change of expression level of mRNA of the integrase gene, detection on microorganism drug resistance, detection on integration efficiency of the integrase and the like, interference efficiency on the siRNA is evaluated, and finally, the siRNA can control and eliminate the drug resistance of pathogenic microorganisms due to the integron. The siRNA can highly-effectively and specifically interfere in the expression of the integrase and the drug-resistance gene so as to inhibit the drug resistance of bacteria, thereby developing a new way for development of novel medicines.

Description

technical field [0001] The invention relates to siRNA for inhibiting microbial drug resistance and application thereof. Background technique [0002] Food safety and food-borne microbial contamination are still ancient and challenging topics in food science. The so-called microbial resistance refers to the resistance to a certain concentration of antibacterial drugs that can usually inhibit their growth and reproduction. This concept was proposed in the 1950s, only 4 years from the clinical application of the first antibiotic, penicillin. Bacteria carrying drug resistance factors can be transmitted among animals, plants, microorganisms and humans through fertilizers, feed, and food, but the transmission of drug resistance to humans through the food chain is considered to be the main cause of drug resistance in human pathogens. Human beings have continuously developed new antibacterial drugs to suppress and eliminate the growing complex drug resistance of bacteria, but new ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P31/00
CPCA61K31/713C12N15/1137C12N2310/141Y02A50/30
Inventor 赵俊仁闫鹤石磊李春海郭先霞
Owner GUANGDONG UNIV OF PETROCHEMICAL TECH
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