Antisense nucleic acid of methicillin-resistant staphylococcus aureus resistance gene mecA

An anti-methicillin-resistant, methicillin-resistant technology, applied in the direction of antibacterial drugs, pharmaceutical formulations, recombinant DNA technology, etc., can solve the problem of antisense nucleic acid deficiency, achieve broad application prospects, and inhibit MRSA drug resistance Effect

Inactive Publication Date: 2012-07-04
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is still no international unified standard for the design of antisense nucleic acid. Usually, people use some computer software to analyze the structure of the target gene for assisted design.

Method used

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  • Antisense nucleic acid of methicillin-resistant staphylococcus aureus resistance gene mecA
  • Antisense nucleic acid of methicillin-resistant staphylococcus aureus resistance gene mecA
  • Antisense nucleic acid of methicillin-resistant staphylococcus aureus resistance gene mecA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Design of antisense thio-oligodeoxynucleotides (PS-ODNs) against mecA mRNA:

[0025]The genome sequence of the MRSA drug-resistant gene mecA (gene number GI: 2791983) was retrieved from GENBANK. The sequence is 2007 bp in length and encodes PBP2a, which determines the expression of MRSA drug resistance. According to the principle of base complementarity, the mRNA of mecA was obtained from the DNA sequence. We use RNAstructure4.2 computer software to assist in the design, screen out a series of antisense nucleic acid target sites, and design corresponding antisense nucleic acid based on these target sites, RNAstructure can predict the secondary structure of genes according to the principle of minimum free energy , calculate the energy change when PS-ODN binds to each target, and according to the net energy (Overall ΔG) of PS-ODN binding to target sequence, the energy required for PS-ODN binding to linear target sequence (Duplex ΔG or Binding ΔG), after PS-ODN-...

Embodiment 2

[0028] Design and synthesis of embodiment 2 PCR primers:

[0029] According to the gene sequence of mecA and 16srRNA of MRSA reported in GeneBank, two pairs of specific amplification primers for mecA and 16srRNA were designed with the software PrimerPremier5.0. The primers were verified to be highly specific by BLAST software, and were synthesized by Shanghai Bioengineering Technology Service Co., Ltd. The sequences are shown in Table 2.

[0030] Table 2 Design and synthesis of amplification primers

[0031]

Embodiment 3

[0032] The preparation of embodiment 3 anionic liposomes:

[0033] First, we prepared positively charged nanoparticles through electrostatic interaction between PEI and PS-ODN. Stearoylphosphatidylethanolamine (PEG 2000 -DSPE), the film dispersion method prepares anionic liposomes as the drug delivery system for transducing PS-ODN into the MRSA bacterial body.

[0034] In the following examples, the applicant selects PS-ODNs09, which has the best experimental results, as an example. Other thio-oligodeoxynucleotides are also tested according to the following methods.

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Abstract

The invention relates to antisense oligodeoxynucleotide sequence aiming at a drug resistant gene mecA of a methicillin resistant staphylococcus aureus (MRSA), preparation for medicines containing the antisense oligodeoxynucleotide sequence, and application of the antisense oligodeoxynucleotide sequence. The antisense oligodeoxynucleotide sequence is characterized in that the antisense oligodeoxynucleotide sequence can be combined with different regions of the drug resistant gene mecA of the MRSA, and block the expression of the drug resistant gene mecA; the base sequence of the antisense oligodeoxynucleotide is PS-ODNs01-20; and the antisense oligodeoxynucleotide can be combined with a specific site of a target gene, so as to inhibit the expression of the drug resistant gene, restore the sensitivity of MRSA to beta-lactam antibiotics, and effectively resist the drug resistance of MRSA.

Description

technical field [0001] The invention relates to a kind of antisense nucleic acid sequence for methicillin-resistant Staphylococcus aureus (MRSA) drug-resistant gene mecA, preparation of medicine containing the antisense nucleic acid sequence and application thereof. Background technique [0002] At present, the problem of bacterial drug resistance has intensified around the world, and many bacterial infectious diseases that were once curable are becoming incurable diseases. MRSA is the most common and important pathogenic bacteria in clinical practice. Since MRSA was first discovered in 1961, especially after the 1980s, the infection rate of MRSA has shown an obvious upward trend in most areas. For example, the infection rate of MRSA in the United States has increased from 1974 to 2% in 2003 to nearly 60% in 2003, an increase of almost 30 times in 30 years; Italy rose from 6% in 1981 to 26% in 1996. With the continuous improvement of MRSA infection rate, its fatality rate i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11A61K31/7088A61P31/04C12N15/113
Inventor 罗晓星孟静茹王慧贾敏侯征马雪何功浩
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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