Riboswitch aac and application thereof in preparing antibiotics

A nuclear switch and antibiotic technology, applied in the biological field, can solve the problem that erythromycin cannot be recombined, and achieve the effect of high sensitivity and universal sensitivity

Inactive Publication Date: 2013-07-03
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, some clinically isolated erythromycin-resistant Staphylococcus aureus encodes a class of erythromycin-resistant RNA methyltransferase (Erythromycin resistance RNA methyltransferase C, ERM C) in the genome, and the ERM protein can Adenine A at position 2058 on the 23S RNA is methylated, and the methylated position A2058 will affect the local conformation of ribosomal RNA, making it impossible for erythromycin to bind to this region, resulting in bacterial drug resistance [The structural basis of ribosome activity in peptide bond synthesis. Nissen, P., Hansen, J.B., N., Moore, P., and Steitz, T., Science, 2000.289(5481): p.920-30.The bacterial ribosome, a promising focus for structure-based drug design. Knowles D, Foloppe N, Matassova N, Murchie A I H. Current Opinion in Pharmacology, 2002.2(5): p.501-6.]

Method used

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  • Riboswitch aac and application thereof in preparing antibiotics
  • Riboswitch aac and application thereof in preparing antibiotics
  • Riboswitch aac and application thereof in preparing antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Reporter gene carrier construction

[0053] According to the fact that aminoglycoside antibiotics are easy to act on the A site of 16S rRNA, a clustwal comparison was performed on the website of the European Biology Center, and the results showed that there are many identical bases between aac and the A site, such asfigure 1 shown.

[0054] In vivo verification test, by digesting plasmid pGEX4T-3 (4968bp) at BspMI and Tth111I, inserting tac promoter (Ptac), lac operator (Olac), aac sequence, lacZα sequence, tryptophan terminator Ttrp sequence, The plasmid was named pGEX-aac-lacZα.

[0055] The resistance gene RmtB was inserted between kasI / NarI and BspMI, and the plasmid was named pGEX-RmtB-aac-lacZα. Such as Figure 2-4 shown.

Embodiment 2

[0056] Example 2: Determination of lacZ enzyme activity

[0057] Transfer 5ml of the overnight cultured JM109 containing the transformed plasmid pGEX-aac-lacZα into fresh LB liquid medium at a ratio of 1:100, add 100μg / ml ampicillin, 0.5mM IPTG, and incubate at 37°C for 1.5-2 hours; branch into sterile glass tube, add different concentrations of aminoglycoside antibiotics, and incubate for 24 hours; measure and record the absorbance of Abs600; draw 20 μl of bacterial liquid and add 80 μl of solution 1 (100 mM Na 2 HPO 4 .20mM KCl, 2mM MgSO 4 .0.8mg / mL CTAB, 0.4mg / mL sodium deoxycholate, 5.4μL / mL beta-mercaptoethanol) into 1.5ml EP tube; incubate at 30°C for 20-30min, and incubate solution 2 containing substrate ONPG under the same conditions (60mM Na 2 HPO 4 .40mM NaH 2 PO 4 .1mg / mL ONPG, 2.7μL / mLβ-mercaptoethanol); add 600μl solution 2 to each EP tube, incubate for 2h; add 700μl stop solution (1M Na 2 CO 3 ), mix well; centrifuge at 12,000 rpm for 5-10 min, and measure...

Embodiment 3

[0059] Embodiment 3: fluorescent quantitative PCR

[0060] 1. Bacterial culture

[0061] For the determination of lacZ enzyme activity, cells were collected by centrifugation after adding different concentrations of antibiotics and incubating for 24 hours.

[0062] 2. Extraction of RNA

[0063] After adding 1ml TRIzol to the sample, place it at room temperature for 5 minutes to fully lyse the sample; if the next step is not performed, the sample can be stored at -70°C for a long time; add 200 μl chloroform, vibrate vigorously and mix well, then place it at room temperature for 3-5 minutes to allow it to separate naturally. phase; centrifuge at 12,000rpm at 4°C for 10-15min. Transfer the aqueous phase (usually 550 μl) to a new tube; add an equal volume of ice-cold isopropanol to the supernatant, and place it at room temperature for 10-20 minutes; centrifuge at 12,000 rpm at 4°C for 10 minutes, discard the supernatant, and precipitate the RNA at the bottom of the tube ;Add 1m...

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Abstract

The invention belongs to the field of biotechnology, relates to a novel antibiotic drug screening target spot, particularly to a sequence and a preparation method of aac riboswitches, and an application of the aac riboswitches as the novel target, antibiotic screening target. The invention provides a riboswitch, of which the DNA sequence is shown as SEQ IN NO 1, wherein N is any one of A, T, G or C; or the DNA sequence of the riboswitch is shown as SEQ IN NO 2. The characters of the riboswitch that the RNA sequence thereof can be combined with an antibiotic easily and further expression of a resistance gene is caused are utilized, so that the riboswitch can serve as a drug target for screening a novel antibiotic or reducing drug resistance of microorganisms. The riboswitch provides a new idea for an inductive drug resistance research.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to an antibiotic and a nuclear switch related to drug resistance. More specifically, the present invention relates to the in vitro transcription of the riboswitch "aac", its structural changes upon interaction with antibiotics, and its use as a target for antibiotic action. Background technique [0002] The discovery of antibiotics has brought good news to humans in the effective treatment of various bacterial infections. However, the widespread use and even misuse of antibiotics has resulted in a huge increase in antibiotic resistance, causing a large number of antibiotics to lose their effect. According to statistics, the number of people who die from various bacterial infections in the world every year is 8 times higher than that of ten years ago. Therefore, it is very important to discover new antibiotics, and the key point is to find new targets of antibiotics. [0003] Ribosome is ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N15/10C12Q1/68C12Q1/02
Inventor 贾旭张静何维志陈东戎阿拉斯太尔·马歇尔
Owner FUDAN UNIV
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