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Antisense peptide nucleic acid of cell penetrating peptide-mediated antibacterial RNA polymerase sigma 70 factor gene rpoD

An RNA polymerase and peptide nucleic acid technology, which is applied in the field of antisense peptide nucleic acid of antibacterial RNA polymerase σ70 factor gene rpoD mediated by permeable peptides, and achieves the effect of broad application prospects.

Inactive Publication Date: 2010-11-24
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The gene sequence of the σ70 factor is highly conserved, except for the non-conserved region between 1.2 and 2.1, the gene sequence of each region rarely mutates, so it is an ideal target for antisense drugs At present, there is no selective action on σ70 factor antibacterial agents

Method used

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  • Antisense peptide nucleic acid of cell penetrating peptide-mediated antibacterial RNA polymerase sigma 70 factor gene rpoD
  • Antisense peptide nucleic acid of cell penetrating peptide-mediated antibacterial RNA polymerase sigma 70 factor gene rpoD
  • Antisense peptide nucleic acid of cell penetrating peptide-mediated antibacterial RNA polymerase sigma 70 factor gene rpoD

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Membrane-penetrating peptide-mediated antibacterial RNA polymerase σ 70 Design of antisense peptide nucleic acid of factor gene rpoD:

[0029] RNA polymerase σ of Escherichia coli str.K-12 substr.MG1655 retrieved in GENBANK 70 The sequence of the factor encoding gene rpoD (gene number gi: 49175990), the full length of the sequence is 1842bp, the effective sequence of RpoD is between 1bp and 613bp of the sequence, of which 1bp to 65bp encodes the 1.1 region of RpoD, and 95bp to 126bp encodes RpoD The 1.2 region of 137bp to 348bp encodes the non-essential region of RpoD, the 379bp to 449bp encodes the 2.0 region of RpoD, the 453bp to 535bp encodes the 3.0 region of RpoD, and the 541bp to 599bp encodes the 4.0 region of RpoD.

[0030] RNA polymerase σ of Salmonella enterica subsp.enterica serovar Paratyphi A str. retrieved in GENBANK 70 The sequence of the factor encoding gene rpoD (gene number gi: 56412276), the full length of the sequence is 1983bp, the effec...

Embodiment 2

[0041] Example 2 Preparation and separation and purification of peptide nucleic acid-penetrating peptide

[0042]In this example, all peptide nucleic acid-penetrating peptide sequences were prepared according to the above sequence, and mismatched and irrelevant peptide nucleic acid-penetrating peptide sequences were prepared as a control. In this example, HATU / DIEA is used as the condensing agent, on MBHA-Rink resin, Fmoc is used to protect the α-amino acid, and the C-terminal is synthesized manually / automatically in solid phase from the C-terminal (carboxyl terminal) to the N-terminal (amino terminal). Amidated peptide nucleic acid-penetrating peptide chain. After the synthesized peptide nucleic acid-penetrating peptide was sheared by high concentration TFA, the crude product was separated and purified by RP-HPLC chromatography, and then the molecular weight of the pure product was determined by MALDI-TOF mass spectrometer.

[0043] The specific test steps are as follows.

...

Embodiment 3

[0049] The MIC determination of embodiment 3 peptide nucleic acid-penetrating peptide

[0050] In the first step, take a sterile 96-well plate, take the non-edge 10 rows × 6 columns = 60 wells, and mark each row as "antibiotic control, peptide nucleic acid-transmembrane peptide 1, 2, 3, 4, 5 ". Add 50 μl of M-H nutrient broth to each well.

[0051] In the second step, 50 μl of antibiotics at a concentration of 2048ug / ml and peptide nucleic acid-penetrating peptides 1, 2, 3, 4, and 5 at a concentration of 100 μM were added to the corresponding marks in the first column of each row.

[0052] Step 3: After mixing well in the first well of each row, aspirate 50 μl and add it to the second well; 50 μl of medicated M-H nutrient broth.

[0053] The fourth step is to pick the single clone of the test strain grown on the M-H agar plate and shake the bacteria in 2ml LB broth to the logarithmic growth phase, draw an appropriate amount of bacterial liquid and dilute it to OD by 2 times...

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Abstract

The invention provides a group of peptide nucleic acid-cell penetrating peptide antisense antibacterial sequences taking bacterial gene rpoD as a target. The cell penetrating peptide-mediated antibacterial RNA polymerase sigma 70 factor gene rpoD which is antisense nucleic acid of a specificity target can selectively inhibit the expression of in-vivo ropD genes of Gram-negative bacterium (containing sensitive and multidrug resisting clinical pathogenicity Escherichia coli, salmonellatyphimurium, Klebsiella pneumoniae and pseudomonas aeruginosa) or gram-positive bacterium (containing sensitiveand multidrug resisting staphylococcus aureus) and further inhibit bacteria growth and reproduction, thus having the advantages of good antibacterial effect, low toxicity, good stability and better tolerance. The invention also discloses a chemical preparation method of a peptide nucleic acid-cell penetrating peptide solid phase. The antibacterial peptide synthesized in the invention can be used for preparing multidrug-resistant bacteria infection proofing antisense drugs and has the potency of being developed into a broad-spectrum antisense antibacterial agent which is expressed by efficiently transferred and peculiarly blocked bacterium disease-causing genes.

Description

technical field [0001] The present invention relates to a group of membrane-penetrating peptide-mediated antibacterial RNA polymerase σ 70 The antisense peptide nucleic acid antibacterial sequence of factor gene rpoD, its preparation method and application in treating multidrug-resistant bacteria infection. Background technique [0002] In the face of the rapid spread of drug-resistant bacteria and the severe situation in which the fatality rate of infectious diseases continues to rise, the control of the harm caused by drug-resistant bacterial infections has become the focus of great attention of governments in various countries. Finding and developing new drugs to effectively control drug-resistant bacterial infections Strategies and new drugs have become an urgent research goal for scientists all over the world. [0003] One of the important strategies of classical antibiotic research and development is to develop new derivatives from various classes of antibiotics. Wit...

Claims

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Application Information

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IPC IPC(8): C07K14/00A61K38/16A61P31/04
CPCY02A50/30
Inventor 罗晓星白卉侯征周颖薛小燕孟静茹桑国军
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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