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  

Screening of effective antisense peptide nucleic acids targeting gyrA from multidrug-resistant Acinetobacter baumannii and their antimicrobial effects in vitro

作　　者：王慧娟[1] 何云燕[2] 夏云[1] 王立朋[1] 梁树梅[1] 

机构地区：[1]重庆医科大学附属第一医院检验科,重庆400016 [2]重庆市中山医院检验科,重庆400014

出　　处：《第三军医大学学报》2013年第14期1442-1446,共5页Journal of Third Military Medical University

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)~~

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。Objective To screen the effective antisense peptide nucleic acids targeting gyrA gene from multidrug-resistant Acinetobacter baumannii, and to evaluate their antimicrobial effects in vitro. Methods Two RNA folding computer programs, Mfold and RNA structure 4.6, were used to predict the secondary structure of gyrA mRNA, and then 10 antisense oligonucleotides were designed based on free energy theory. The full length of gyrA mRNA was transcribed in vitro and labeled by digoxigenin-ll-uridine-5＇-triphosphate. Dot blot hybridization was used to screen the gyrA mRNA accessible sites which showed strong binding affinity to the antisense oligonucleotides. Peptide nucleic acid （PNA） was synthesized based on the sequence of antisense oligonucleotide showing high affinity. Another PNA oligomer containing 6 mismatched nucleotides was used as a negative control. Both the 2 PNAs were conjugated peptide-PNA （PPNA）. After the bacterial culture was viable cell counts were measured to evaluate the to cell penetrating peptide （CPPs） （KFF）3 K to form treated with different concentrations of PPNA, OD60o and growth inhibitory effect of the antisense oligonucleotide. Reverse transcript （RT） -PCR was applied to evaluate the level of gyrA expression. Results Of the 10 antisense oligonucleotides, 5 showed binding affinity to gyrA mRNA and one of them showed strong binding affinity.PPNA designed based on the oligonucleotide significantly inhibited the growth of the bacterium and gyrA gene expression at a dose of 5 μmol/L, and exhibited anti-bactericidal effect at a dose of 10 μmol/L. Mismatched PPNA had no effect on the bacterial growth. Conclusion Combination of computer-aided prediction with dot blot hybridization is a high-flux and rapid way to screen effective antisense oligonucleotides in vitro. The screened anti-gyrA PPNA exerts significant inhibitory effect on the growth and gene expression in the bacterium in vitro.

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 

分 类 号：R378[医药卫生—病原生物学] R394.6[医药卫生—基础医学]