铜绿假单胞菌lasR基因反义肽核酸序列筛选及其抑制生物被膜形成的研究  

Screening of antisense peptide nucleic acid sequence targeting lasR gene of Pseudomonas aeruginosa and its inhibition on biofilm formation

作　　者：张峰领[1] 安琳 李进[1] 李发科[1] 徐欢[1] 罗杰[1] 杨成[1] 蒋文斌[1] 雷国勤 汪超[1] 周琳[1] 鲁卫平[1] Zhang Fengling An Lin Li Jin Li Fake Xu Huan Luo Jie Yang Cheng Jiang Wenbin Lei Guoqin Wang Chao Zhou Lin Lu Weiping(Department of Clinical Laboratory, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 400042 Out-patient Department , Military Representative Bureau of Army, Chongqing, 400060, China)

机构地区：[1]第三军医大学大坪医院野战外科研究所检验科,重庆400042 [2]陆军重庆军事代表局门诊部,重庆400060

出　　处：《第三军医大学学报》2017年第6期529-535,共7页Journal of Third Military Medical University

基　　金：全军医学科研"十二五"计划面上项目(CWS13J039)~~

摘　　要：目的筛选靶向铜绿假单胞菌lasR基因的反义肽核酸序列,探讨其对铜绿假单胞菌的生物被膜形成能力的影响。方法针对铜绿假单胞菌lasR基因设计4条反义寡核苷酸序列,利用斑点杂交筛选出与lasR基因结合最佳的反义寡核苷酸序列,以此合成与穿膜肽连接的肽-肽核酸序列。分别用不同浓度的肽-肽核酸导入铜绿假单胞菌生物被膜模式菌PAO1中,测定不同时相点细菌生长光密度[D(600)]值并进行菌落计数,观察肽-肽核酸对其生长的抑制作用。利用光学显微镜和扫描电镜观察生物被膜形成情况。q PCR方法检测lasR mRNA表达。结果斑点杂交实验结果显示:4条反义寡核苷酸序列中3条有杂交信号产生,其中第1条序列信号最强,以此为基础合成反义肽核酸。不同浓度的肽-肽核酸对铜绿假单胞菌表现出不同程度的体外抗菌活性,且随着浓度增加,抗菌活性增强。结论有效筛选出高效反义核苷酸序列,经筛选出的靶向铜绿假单胞菌lasR基因的反义肽核酸对铜绿假单胞菌的生长及生物被膜形成能力有明显抑制作用,同时抑制lasR mRNA的表达。Objective To screen antisense peptide nucleic acid sequence targeting lasR gene of Pseudomonas aeruginosa（ PA） and investigate its inhibitory effect on biofilm formation. Methods Based on the sequence of lasR gene of PA,4 types of antisense nucleic acid sequences were designed,and based on the principle of dot blot,1 antisense nucleic acid sequence which could well bind with lasR gene was screened.Subsequently,peptide-peptide nucleic acids were synthesized and bound with penetrating peptides. Different concentrations of peptide-peptide nucleic acids were introduced into PAO1（ biofilm model strain of PA）,D（ 600） value and plate count were measured for the inhibitory eftect of the peptide-peptide nuclear acids on the growth of PA. The biofilm formation was observed by light microscopy and scanning electron microscopy.LasR mRNA expression was detected with quantitative real-time polymerase chain reaction（ q PCR）.Results Three types of the 4 antisense oligonucleotides showed binding affinity to lasR mRNA at dot spot hybridization experiment. The first strip antisense oligonucleotide presented strongest singal,and an antisense nucleic acid sequence was synthesized based on the result. The antimicrobial activity was enhanced with increase of the concentration of peptide-peptide nucleic acids. Conclusion The antisense peptide nucleic acid sequence targeting lasR gene of PA has significant inhibitory effect on the growth and biofilm formation of PA,and suppresses the expression of lasR mRNA.

关 键 词：铜绿假单胞菌 lasR基因 反义肽核酸 生物被膜 

分 类 号：R379.991[医药卫生—病原生物学] R394-33[医药卫生—基础医学]