体外斑点杂交筛选靶向多重耐药鲍曼不动杆菌高效反义核酸序列实验研究  

Screening of effective antisense nucleotides targeting multidrug resistant Acinetobacter baumannii by in vitro dot spot hybridization

作　　者：梁树梅[1] 何云燕[2] 夏云[1] 王慧娟[1] 王立朋[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：《中国抗生素杂志》2014年第8期620-624,共5页Chinese Journal of Antibiotics

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：目的体外斑点杂交实验模拟体内环境验证计算机辅助软件设计的反义寡核苷酸序列的活性。方法采用计算机辅助软件对多重耐药鲍曼不动杆菌生长必须基因gyrA的mRNA进行二级结构分析计算,设计出10条反义寡核苷酸序列,合成探针与体外转录并用地高辛标记的gyrA mRNA进行斑点杂交,分别选择一条杂交信号最强和一条杂交信号弱的探针序列合成连接穿膜肽序列的肽-肽核酸,以不同浓度的肽-肽核酸处理鲍曼不动杆菌标准株ATCC19606和多重耐药株,测定经不同浓度PPNA处理的细菌光密度A600值并进行平板菌落计数,观察其对细菌生长的抑制作用。以20μmol/L杂交信号强的肽-肽核酸作用于铜绿假单胞菌,测细菌光密度A600值,观察其对细菌生长的抑制作用。结果杂交信号强的肽-肽核酸对多重耐药的鲍曼不动杆菌和ATCC19606均有明显的体外抗菌活性,其最低抑菌浓度分别为5与10μmol/L,10μmol/L浓度时对两种细菌均具有杀菌活性,而杂交信号弱的肽-肽核酸对细菌的生长无抑制作用,即使将其浓度增加到20μmol/L。杂交信号强的肽-肽核酸对铜绿假单胞菌无生长抑制作用。结论体外斑点杂交实验能够验证计算机软件预测设计的具有特异性的反义核酸的活性,为体外可靠、快速、高通量筛选高效反义序列提供了一种有效的方法,并且为筛选所有基因高效反义序列搭建了一个平台。Objective To verify the activity of antisense oligonucleotides designed by computer software by using dot spot hybridization which is simulated the environment in vivo. Methods gyrA mRNA optimal secondary structure regions were predicted by computer program and 10 potential antisense oligonucleotidesprobes were designed. The full length of gyrA mRNA transcribed and labeled by digoxigenin-11-uridine-5＇-triphosphate was hybridized with the designed antisense oligonucleotides by dot blot hybridization. The probe with the strongest hybridization signal density and one of the weak hybridization signal density were selected respectively as the sequences for PNA synthesizing. Both of the 2 PNAs were conjugated to cell penetrating peptide （CPPs） （ KFF）3 K to form PPNA. Acinetobacter baumannii standard strain ATCC19606 and multi-drug resistant strain CY-26 were treated with different concentrations of PPNA. A600 and viable cell counts were measured to evaluate the inhibitory and bactericidal effect of the antisense oligonucleotide. And Pseudomonas aeruginosa were treated with PPNA which is a probe with the strongest hybridization signal density. Results The probe with the strongest hybridization signal density can totally inhibit the growth of the multi-resistance Acinetobacter baumannii at the concentration of 5 ~tmol/L, inhibit the growth of ATCC 19606 at the concentration of 10~tmol/L, and exhibit bactericidal effect in two strains of Acinetobacter baumannii at a concentration of 10μmol/L, the PPNA with weak hybridization signal density exhibited no growth inhibitory effect at higher concentrations. Even the concentration of PPNA increased to 20μmol/L, and the PPNA with the strongest hybridization signal density exhibited no growth inhibitory effect to Pseudomonas aeruginosa at concentration of 20μmol/L. Conclusion Dot spot hybridization can verify the activity of the sequence specificity of antisense nucleotide designed by computer software, which is a reliable, high-flux and rapid way to screen effective

关 键 词：鲍曼不动杆菌 GYRA 斑点杂交 反义寡核苷酸 肽核酸 

分 类 号：R378[医药卫生—病原生物学]