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SiRNA for regulating expression of integrase and drug-resistant gene cassette and application thereof

A drug-resistant gene, integrase technology, applied in DNA/RNA fragments, gene therapy, genetic engineering, etc., can solve problems such as gene-specific silencing

Pending Publication Date: 2017-04-19
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 regulating expression of integrase and drug-resistant gene cassette and application thereof
  • SiRNA for regulating expression of integrase and drug-resistant gene cassette and application thereof
  • SiRNA for regulating expression of integrase and drug-resistant gene cassette and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, siRNA

[0028] Through a large number of experimental studies and screenings in the early stage, the present invention finally found that two kinds of siRNAs have good effects on the capture of drug-resistant gene cassettes and the expression of drug-resistant genes by integrase genes in silencing and interfering bacterial integrin systems (high efficiency and specificity) ), the specific sequences of these two siRNAs are as follows.

[0029] siRNA-I:

[0030] siRNA-1 sense strand: 5'-GCUGAAAGGUCUGGUCAUATT-3' (SEQ ID NO: 1),

[0031] siRNA-1 antisense strand: 5'-UAUGACCAGACCUUUCAGCTT-3' (SEQ ID NO: 2),

[0032] siRNA-II:

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

[0034] siRNA-II antisense strand: 5'-AGCUUCUGUAUGGAACGGGTT-3' (SEQ ID NO: 4).

[0035]The synthesis of the above siRNA can be directly synthesized by chemical method (can be synthesized by the corresponding biological company). The following methods are ado...

Embodiment 2

[0036] The construction of embodiment 2 siRNA interference carrier

[0037] 1) siRNA annealing

[0038] Dissolve the synthesized siRNA sense strand and antisense strand in TE into a solution with a concentration of 100 μM, and prepare an annealing reaction system according to the following table.

[0039]

[0040] 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.

[0041] 2) Construction of pGPU6 / GFP / Neo-siRNA expression vector

[0042] 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 BbsI and BamHI to obtain the target linear fragment, and then the shRNA fragment obtained above was ligated with the digested pGPU6 / GFP / Neo. The ligation system was as follows:

[0043]

[0044]

[0045] After reacting at 22°C for 1 hour,...

Embodiment 3

[0046] The detection of embodiment 3 siRNA interference efficiency

[0047] experimental method:

[0048] In order to detect the interference efficiency of the siRNA of the present invention, the siRNA-I and siRNA-II interference vectors prepared in Example 2 were transfected into drug-resistant bacteria Escherichia coli E.coli BL21 (DE3), and the drug-resistant E. coli contained the integrated The enzyme is IntI1, and the drug resistance gene cassette contained 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.

[0049] The above RT-PCR system and program are:

[0050] Use Bio-Bad CFX...

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Abstract

The invention discloses an siRNA for regulating expression of integrase and a drug-resistant gene cassette and an application thereof. A drug-resistant bacteria strain is introduced into the designed siRNA, and capture of an integrase gene on the drug-resistant gene cassette and expression of a drug-resistant gene in an integrated subsystem are silenced and interfered. Through RT-PCR quantitative detection of changes of the integrase gene mRNA expression level, microbial drug-resistant detection, integrase integration efficiency detection and other methods, the interference efficiency on the siRNA is evaluated, and the pathogenic microorganism drug-resistant problem caused by integron is controlled and eliminated by the siRNA ultimately. The expression of the integrase and the drug-resistant gene can be effectively and specifically interfered to inhibit bacterial drug resistance, and a new idea is widened for development of new drugs.

Description

technical field [0001] The invention relates to an expression regulation factor siRNA for integrase and drug resistance gene cassette 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 comple...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61P31/04
CPCA61K31/713C12N9/00C12N15/1137C12N2310/141C12N2320/30Y02A50/30
Inventor 赵俊仁闫鹤石磊李春海郭先霞
Owner GUANGDONG UNIV OF PETROCHEMICAL TECH
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