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

发布时间：2018-03-12 11:57

本文选题：铜绿假单胞菌　切入点：lasR基因　出处：《第三军医大学学报》2017年06期 　论文类型：期刊论文

【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。
[Abstract]:Objective to screen the antisense peptide nucleotide sequences of lasR gene targeting Pseudomonas aeruginosa and to explore its effect on the biofilm formation ability of Pseudomonas aeruginosa. Methods four antisense oligonucleotide sequences were designed for lasR gene of Pseudomonas aeruginosa. The best antisense oligonucleotide sequence combined with lasR gene was selected by dot blot hybridization to synthesize the peptide peptide nucleotide sequence which was linked to the transmembrane peptide. Different concentrations of peptide peptide nucleic acid were introduced into PAO1 of Pseudomonas aeruginosa biofilm. The optical density of bacterial growth at different phase points [DX600] was measured and the colony count was carried out. The inhibitory effect of peptide-peptide nucleic acid on its growth was observed. The expression of lasR mRNA was detected by optical microscope and scanning electron microscope. The expression of lasR mRNA was detected by using the method of .q PCR. Results the results of dot blot hybridization showed that there were 4 antisense oligonucleotides. Three of the sequences produced hybrid signals. The antisense peptide nucleic acid was synthesized on the basis of the strongest signal of the first sequence. Different concentrations of peptide-peptide nucleic acid showed different degrees of antibacterial activity against Pseudomonas aeruginosa in vitro, and with the increase of concentration, Conclusion the antisense peptide nucleotides targeting lasR gene of Pseudomonas aeruginosa can inhibit the growth of Pseudomonas aeruginosa and the ability of biofilm formation. The expression of lasR mRNA was also inhibited.
【作者单位】：第三军医大学大坪医院野战外科研究所检验科;陆军重庆军事代表局门诊部;
【基金】：全军医学科研“十二五”计划面上项目(CWS13J039)~~
【分类号】：R378.991

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