多重耐药鲍曼不动杆菌gyrA基因高效反义肽核酸序列筛选及其体外抗菌活性观察

发布时间：2018-02-04 17:14

本文关键词： 多重耐药鲍曼不动杆菌 gyrA 反义寡核苷酸斑点杂交肽核酸　出处：《第三军医大学学报》2013年14期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to screen the antisense peptide nucleic acid (ASN) sequence which can bind to the gyrA gene of Acinetobacter baumannii and evaluate its antibacterial activity in vitro. Methods Mfold was used. The secondary structure of gyrA gene of Acinetobacter baumannii was analyzed and calculated by two kinds of computer software RNA Structure 4.6. According to the principle of minimum free energy, antisense oligonucleotides were designed in the local hybridization relaxation region of mRNA, and dot-hybridized with digoxigenin-labeled gyrAmRNA in vitro. The antisense oligodeoxynucleotides combined with gyrA mRNA were screened according to the intensity of hybridization signal, and the peptide nucleic acid pep-tide nucleic acid was synthesized according to the sequence of antisense oligodeoxynucleotides. A PNA with 6 base mismatches was added as a negative control group, and 2 PNA were connected with peptide-PNA respectively. Determination of the optical density of bacteria treated with different concentrations of PPNA. [The expression of gyrA mRNA was detected by RT-PCR. The results of dot blot hybridization showed that the expression level of gyrA mRNA was detected by dot blot hybridization. Five of the 10 antisense oligonucleotide probes designed by computer software showed hybridization signals with gyrA mRNA, one of which had the strongest signal and could bind stably with gyrA mRNA. The results showed that it could completely inhibit the expression of gyrA mRNA and inhibit the growth of Acinetobacter baumannii at the concentration of 5 渭 mol/L. It has bactericidal activity at the concentration of 10 渭 mol/L. But PPNA with mismatched base can not inhibit the growth of bacteria. Conclusion Computer-aided design combined with dot blot can be used to verify the efficiency of oligonucleotide binding to target gene in vitro. High throughput screening of high efficiency antisense sequences was achieved in vitro. The antisense peptide-peptide nucleic acid targeting gyrA gene could effectively inhibit the growth of Acinetobacter baumannii in vitro.
【作者单位】：重庆医科大学附属第一医院检验科;重庆市中山医院检验科;
【基金】：国家自然科学基金面上项目(81071400) 重庆市自然科学基金面上项目(CSTC2010BB5343) 国家临床重点专科建设项目经费资助([2010]305)~~
【分类号】：R378
【正文快照】： 鲍曼不动杆菌(Acinetobacter baumannii,AB)是引起医院感染最常见的条件致病菌之一,具有极强的环境适应能力和广泛的耐药性。近年来其感染率和耐药性明显上升,尤其是多重耐药(multidrug-resistant,MDR)甚至泛耐药(pan-resistant,PDR)鲍曼不动杆菌的不断出现,并在全球多个国家

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