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
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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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