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Transmembrane peptide-mediated antisense antibacterial agent and preparation method and application thereof

A technology of membrane-penetrating peptide and antibacterial agent, which is applied in the field of antisense antibacterial agent and its preparation, and can solve the problems of difficult penetration and low intracellular concentration

Inactive Publication Date: 2014-06-18
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the prerequisite for LNA to fully exert its antisense effect is to reach an effective concentration at the target mRNA binding site in the bacterial cell, and LNA is a negatively charged macromolecular compound, which is not easy to penetrate the bacterial cell wall and cell membrane, and its intracellular concentration is low. , and can be taken up non-specifically by body cells, so highly efficient, specific bacteria-targeted drug delivery carriers are the bottleneck problem that limits the development of antisense antibacterial agents

Method used

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  • Transmembrane peptide-mediated antisense antibacterial agent and preparation method and application thereof
  • Transmembrane peptide-mediated antisense antibacterial agent and preparation method and application thereof
  • Transmembrane peptide-mediated antisense antibacterial agent and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 designs, synthesizes and screens the LNA of anti-Staphylococcus aureus ftsZ mRNA

[0031] (1) Screen the effective targets of ftsZ mRNA and design specific and efficient LNA: Use RNA Structure4.5 software to analyze the sequence of ftsZ mRNA in detail and predict its secondary structure. The basic length of LNA is 18-21 bases. Conserved sequence regions and non-folding regions select effective targets, and screen out the best 10 LNAs (see Table 1).

[0032] Table 1 Locked nucleic acid (LNA) designed for ftsZ mRNA

[0033]

[0034]Note: The uppercase letters in the nucleic acid sequence column represent LNA, and the lowercase letters represent DNA.

Embodiment 2

[0035] Preparation and separation and purification of embodiment 2 PLNA

[0036] (1) Preparation of membrane-permeable peptide (KFF) 3K: synthesized by fluorenylmethoxycarbonyl solid-phase synthesis method (Fmoc-SPPS), that is, using HATU / DIEA as a condensation agent, on MBHA-Rink resin, using Fmoc to protect α- Amino acids, from the C-terminal (carboxyl-terminal) to the N-terminal (amino-terminal) solid-phase synthesis of polypeptides with cysteine ​​at the C-terminal, the synthesized polypeptides are cleaved by high-concentration TFA and separated and purified by RP-HPLC. The amino acid sequence of the permeable peptide (KFF) 3K is KFFKFFKFFK, K is lysine (Lys), F is phenylalanine (Phe).

[0037] The specific steps are: the first step, solid-phase synthesis of polypeptides: the prepared polypeptides are carried out one by one from the C-terminus to the N-terminus. First, MBHA-Rink resin is packed into a column, and dichloromethane (CHCl 2 ), make it swell, deprotect with 20...

Embodiment 3

[0044] Embodiment 3 PLNA studies on the minimum inhibitory concentration of Mu50

[0045] According to the method of Example 2, it is combined with the permeable peptide (KFF) which promotes LNA to pass through the cell wall at the carbon end of LNA respectively. 3 K phase connection, prepared into PLNA.

[0046] Combining improved microplate MIC determination method and colony counting method as a method for effective antibacterial antisense target screening. The inventor first determined the MIC of 10 anti-ftsZ PLNA sequences (anti-ftsZ PLNA 01, 201, 274, 309, 322, 571, 593, 705, 787 and 1153) to S. aureus (see Table 2), and the results It showed that all 10 PLNAs had different antibacterial activities. Each 10 μM PLNA was co-incubated with Mu50 for 8 hours, and the plate cloning experiment was carried out, and the CFU was counted. The results showed that 10 PLNAs could significantly reduce the number of Mu50 colonies ( figure 2 ). Based on the experimental results of t...

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Abstract

The invention discloses a transmembrane peptide-mediated antisense antibacterial agent which comprises popliteal lymph node assay (PLNA) 787; and the PLNA 787 is formed by connecting transmembrane peptide (KFF) 3K and LNA 787 by cysteine-(4-(N-maleimide methyl)-1- cyclohexidine actidione)-hexane and is prepared by a solid phase synthesis method. The transmembrane peptide-mediated antisense antibacterial agent takes bacteria curing gene as a target spot, has the advantages of being high in specificity, efficient, low in toxicity, safe and the like, has remarkable inhibition effect on the growth of drug-resistant bacteria, and especially has remarkable antagonism on the growth of methicillin-resistant staphylococcus aureus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an antisense antibacterial agent mediated by a membrane-penetrating peptide and its preparation method and application. Background technique [0002] Staphylococcus aureus is the most common and important pathogenic bacteria in hospital-acquired infection and community-acquired infection. With the widespread use of antibiotics, the drug resistance of Staphylococcus aureus is becoming more and more serious. At present, more than 90% of S. aureus are resistant to penicillin. Since the first report of methicillin-resistant staphylococcus aureus (MRSA) in the United Kingdom in 1961, infections caused by MRSA have spread all over the world, and its detection The output rate keeps increasing. MRSA is resistant to all β-lactam antibiotics and other antibacterial drugs, so the infection caused by MRSA is very difficult to treat and has a high mortality rate. At pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06A61K48/00A61K47/48A61P31/04
Inventor 罗晓星孟静茹马雪薛小燕贾敏侯征达飞桑国军
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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