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Application of antisense deoxyribozyme capable of resisting agr quorum-sensing system in MRSA (Methicillin-Resistant Staphylococcus Aureus)

A deoxyribozyme and antisense technology, applied in the field of molecular biology, to achieve the effect of preventing the increase of bacterial drug resistance, weakening bacterial virulence, and reducing secretion

Inactive Publication Date: 2015-01-21
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most effective drug for treating MRSA is vancomycin. However, with the emergence of vancomycin-resistant MRSA, people are about to face a situation where no drug is available. Research Hotspots and Difficulties in the Field of Infection Therapy

Method used

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  • Application of antisense deoxyribozyme capable of resisting agr quorum-sensing system in MRSA (Methicillin-Resistant Staphylococcus Aureus)
  • Application of antisense deoxyribozyme capable of resisting agr quorum-sensing system in MRSA (Methicillin-Resistant Staphylococcus Aureus)
  • Application of antisense deoxyribozyme capable of resisting agr quorum-sensing system in MRSA (Methicillin-Resistant Staphylococcus Aureus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Design, synthesis and screening of DNAzyme against Staphylococcus aureus RNAIII mRNA

[0018] (1) Screen the effective targets of RNAIII mRNA and design specific and efficient DNAzyme: use RNA Structure4.5 software to analyze the sequence of RNAIII mRNA in detail and predict its secondary structure, with 30 bases as the basic length of DNAzyme, in the non-conserved sequence region, Effective targets were selected in the non-folding region, and seven optimal DNAzymes were screened out (see Table 1).

[0019] Table 1 DNAzymes designed for RNAIII mRNA

[0020]

[0021] Note: The underline in the sequence column represents the catalytic active center of DNAzyme.

Embodiment 2

[0022] Example 2 DNAzyme study on the minimum inhibitory concentration of LAC

[0023] The micro-plate MIC assay method is used as a screening method for effective antibacterial antisense targets. The inventors first measured the MIC of 7 anti-RNAIII DNAzyme sequences (anti-RNAIII DNAzyme 23, 70, 72, 105, 109, 232-1 and 232-2) to LAC, and the results showed that none of the 7 DNAzymes had antibacterial activity ( See Table 2).

[0024] Table 2 The minimum inhibitory concentration (MIC) of DNAzyme to LAC

[0025]

Embodiment 3

[0026] Example 3 RT-PCR detection of the influence of DNAzyme on the expression changes of RNAIII

[0027] The fresh overnight cultured bacterial solution was transferred to nutrient broth medium, cultivated at 35°C and 210rpm to the mid-logarithmic growth phase, and DNAzyme23 of different concentrations was added to the culture medium, and incubated for 6 hours. After the culture, the bacteria were collected, the total RNA of the bacteria was extracted by the Trizol method, and the cDNA obtained by reverse transcription was used as a reaction template. The RT reaction conditions were: 37 ° C, 60 min, and PCR was performed after ice bathing. PCR reaction conditions: pre-denaturation at 95°C for 2min, denaturation at 95°C for 30sec, annealing at 55°C for 30sec, extension at 72°C for 30sec, 40 cycles. PCR primers: 5'-TTCAATGGCACAAGAT-3' and 5'-GTCCAAGGAAACTAAC-3'. figure 1 The results showed that the three sequences of DNAzyme23, DNAzyme72 and DNAzyme232-1 among the seven anti...

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Abstract

The invention discloses an antisense deoxyribozyme-DNAzyme23. The antisense deoxyribozyme disclosed by the invention adopts an effector molecule RNAIII in the staphylococcus aureus agr quorum-sensing system as a target spot, does not affect the growth state of the bacterium, can effectively block an agr signaling pathway, can significantly weaken the bacterial virulence and particularly can significantly reduce the pathogenicity of methicillin-resistant staphylococcus aureus. The antisense deoxyribozyme has the advantages of high specificity, low toxicity, safety and difficulty in inducing the increase of bacterial drug resistance and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to an antisense antibacterial deoxyribozyme for inhibiting MRSA quorum sensing agr system and its preparation method and application. Background technique [0002] Staphylococcus aureus is closely related to a variety of human infectious diseases, such as skin infections, respiratory infections, and even life-threatening necrotizing myositis and necrotizing pneumonia. At present, the most effective drug for treating MRSA is vancomycin. However, with the emergence of vancomycin-resistant MRSA, people are about to face a situation where no drug is available. Research hotspots and difficulties in the field of infection treatment. [0003] The abuse of antibiotics by humans is one of the most important reasons for bacterial drug resistance. Under the continuous selective pressure of antibiotics, bacterial drug resistance is an inevitable event. Once each new antibiotic comes ou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P31/04
Inventor 侯征罗晓星薛小燕李明凯周颖孟静茹达飞
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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