耐甲氧西林金黄色葡萄球菌ftsZ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 highly effective antisense peptide nucleic acids targeting the ftsZ gene of methicillin-resistant Staphylococcus aureus and evaluate its antibacterial activity in vitro

作　　者：梁树梅 杨莉莉[1] 刘华伟[1] 夏云[2] Liang Shu-mei;Yang Li-li;Liu Hua-wei;Xia yun(Department of Clinical Laboratory,the Third People's Hospital of Chengdu,Chengdu 610031;Department of Laboratory Medicine,the First Affiliated Hospital of Chongqing Medical University,Chongqing 400016)

机构地区：[1]成都市第三人民医院,成都610031 [2]重庆医科大学附属第一医院检验科,重庆400016

出　　处：《中国抗生素杂志》2020年第9期867-872,共6页Chinese Journal of Antibiotics

摘　　要：目的筛选出能与耐甲氧西林金黄色葡萄球菌ftsZ基因紧密结合的反义肽核酸序列,评价其体外抗菌活性。方法选用两种计算机软件(Mfold、RNA Structure 5.2)和斑点杂交筛选出最佳反义序列PNA,并连接穿膜肽(cell penetrating peptide,CCPs)形成肽-PNA(peptide-PNA,PPNA),另设一条与最好序列有3个错配碱基的肽核酸序列作为对照,连接穿肽形成错配的肽肽核酸(scrambled peptide-PNA,Scr PPNA)。将不同浓度的肽肽核酸处理MRSA后,间隔1h测定A600值同时做平板菌落计数,观测其对细菌生长的抑制作用。结果斑点杂交实验结果表明,计算机辅助软件设计的11条反义寡核苷酸序列中,有4条序列显示强弱不同的杂交信号,第3号探针杂交信号最强,而1条正义序列没显示任何信号,PPNA在30和40μmol/L分别具有抑菌作用和杀菌作用,并具有浓度依赖性,而Scr PPNA在高浓度时对细菌也无生长抑制作用,验证了肽核酸的特异性。结论成功的筛选出一条在体外对MRSA的生长有显著的影响的反义肽核酸序列,有望为抗MRSA感染患者提供新的治疗方案。Objective To screen the antisense oligonucleotides targeting the ftsZ mRNA of methicillinresistant Staphylococcus aureus(MRSA)and evaluate their antibacterial activity in vitro.Methods Antisense peptide nucleic acid(PNA)were identified with computational prediction(Mfold and RNA structure 5.2)and dot-blot hybridization.Scrambled PNA was constructed with mismatches to three bases within the antisense PNA sequence.These two antisense peptide nucleic acids(PNAs)were conjugated to a cell-penetrating peptide.Methicillin-resistant Staphylococcus aureus(MRSA)were treated with different concentrations.The optical density at 600nm(A600)and viable cell counts every hour were measured to the growth rates of MRSA.Results Only four oligonucleotides exhibited different levels of hybridization signals on the dot blot.Of these,the number 3 probe showed the strongest hybridization signal,whereas the negative control(with a sense sequence)showed no hybridized signal.PPNA had bacteriostatic and bactericidal effects at 30 and 40μmol/L,respectively.The scrambled PPNA had no growth inhibition effects,even at higher concentrations,confirming the sequence specificity of the probes.Conclusion We successfully screened a antisense peptide nucleic acid sequence which was expected to become a new therapeutic approach.

关 键 词：耐甲氧西林金黄色葡萄球菌 FTSZ 义寡核苷酸 斑点杂交 

分 类 号：R978.1[医药卫生—药品]