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目的筛选出能与多重耐药鲍曼不动杆菌gyrA基因的mRNA紧密结合的反义肽核酸序列,评估其体外抗菌活性。方法利用Mfold、RNA Structure 4.6两种计算机软件对多重耐药鲍曼不动杆菌gyrA基因的mRNA进行二级结构分析计算,根据最低自由能原则在其mRNA局部杂交松弛区设计反义寡核苷酸,与体外转录的地高辛标记的gyrAmRNA进行斑点杂交,根据杂交信号的强弱筛选出与gyrA mRNA紧密结合的反义寡核苷酸,根据其序列合成肽核酸(pep-tide nucleic acid,PNA),另加设1条与靶序列有6个碱基错配的PNA作为阴性对照,2条PNA分别连接穿膜肽形成肽-肽核酸(peptide-PNA,PPNA)。测定经不同浓度PPNA处理的细菌光密度[D(600)]值并进行平板菌落计数,观察其对细菌生长的抑制作用,采用RT-PCR检测gyrA mRNA的表达水平。结果斑点杂交结果显示,计算机软件设计的10条反义寡核苷酸探针中有5条与gyrA mRNA显示出杂交信号,其中1条信号最强,能够与gyrA mRNA稳定结合,将其以肽-肽核酸的形式合成处理细菌,结果表明其在5μmol/L浓度可完全抑制gyrA mRNA的表达,并抑制鲍曼不动杆菌的生长,在10μmol/L浓度具有杀菌活性,而具有错配碱基的PPNA对细菌生长无明显抑制作用。结论计算机辅助设计联合斑点杂交可在体外模拟体内环境对寡核苷酸与靶基因结合的有效性进行验证,实现在体外高通量筛选高效反义序列。经筛选得到的靶向gyrA基因反义肽-肽核酸可在体外高效抑制多重耐药鲍曼不动杆菌生长。
Objective To screen the antisense peptide sequence which binds closely to the mRNA of gyrA gene of multidrug-resistant Acinetobacter baumannii and to evaluate its antimicrobial activity in vitro. Methods The secondary structure of gyrA gene of multidrug-resistant Acinetobacter baumannii was analyzed by Mfold and RNA Structure 4.6 softwares. Based on the lowest free energy principle, antisense oligodeoxynucleotides , Hybridized with digoxigenin-labeled gyrAmRNA in vitro, antisense oligonucleotides that tightly bind to gyrA mRNA were screened according to the intensity of the hybridization signal, and pep-tide nucleic acid (PNA) ), Plus one additional PNA with a 6 base mismatch with the target sequence as a negative control. Two PNAs were each linked to a transmembrane peptide to form a peptide-peptide nucleic acid (PPNA). The optical density [D (600)] of the bacteria treated with different concentrations of PPNA was measured and the plate colonies were counted to observe the inhibitory effect on the growth of the bacteria. The gyrA mRNA expression level was detected by RT-PCR. Results The results of dot blot showed that 5 of the 10 antisense oligonucleotide probes designed by computer software showed hybridization signals with gyrA mRNA, of which 1 signal was the strongest and could stably bind with gyrA mRNA, - peptide nucleic acids form the synthesis of bacteria, the results showed that its 5μmol / L concentration can completely inhibit gyrA mRNA expression and inhibit the growth of Acinetobacter baumannii in 10μmol / L concentration with bactericidal activity, with mismatched base PPNA had no significant inhibitory effect on bacterial growth. Conclusion The combination of CAD and dot blot can validate the binding of oligonucleotides to target genes in vitro and achieve high-throughput screening of high-efficiency antisense sequences in vitro. The screened gyrA gene antisense peptide - peptide nucleic acid can effectively inhibit the growth of multidrug resistant Acinetobacter baumannii in vitro.