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目的研究肽核酸(peptide nucleic acid,PNA)对铜绿假单胞菌生物膜形成的抑制作用,探讨其潜在的抗铜绿假单胞菌生物膜形成的应用价值。方法采用计算机软件设计和斑点杂交联合筛选出能与铜绿假单胞菌PAO1早期生物膜形成关键基因motA mRNA结合紧密的反义寡核苷酸序列,根据其序列合成肽核酸(PNA)并连接穿膜肽(cell pene-trating peptide,CCPs)(KFF)3K形成CCPs-PNA,分别采用不同浓度的CCPs、PNA和CCPs-PNA处理铜绿假单胞菌PAO1,定量测定并用显微镜观察其对生物膜形成的抑制作用。结果 PNA和CCPs-PNA对铜绿假单胞菌PAO1生物膜的早期形成都有抑制作用,并且随着浓度的增加,其抑制作用也增加,但CCPs-PNA对生物膜形成的抑制明显优于PNA组。1、5、10μmol/L的(KFF)3K-PNA对PAO1生物膜形成的抑制率分别约为30%、50%、70%。而1μmol/L的PNA对PAO1生物膜形成基本没有影响,5、10μmol/L的PNA对PAO1生物膜形成的抑制率却分别为3%和10%左右。各个浓度的CCPs对细菌生物膜的形成都没有影响。与对照组相比,CCPs-PNA处理之后的PAO1,其运动能力明显下降,PAO1处理组与对照组在运动培养基上的扩散直径之比约为3∶5。结论针对motA基因的PNA通过抑制PAO1的运动能力、降低吸附,从而对铜绿假单胞菌PA01起始阶段的生物膜形成产生抑制作用,穿膜肽(KFF)3K大大增强了此作用。推测铜绿假单胞菌的motA基因或其编码产物可能是一个良好的抗铜绿假单胞菌生物膜形成的靶点。
Objective To study the inhibitory effect of peptide nucleic acid (PNA) on the biofilm formation of Pseudomonas aeruginosa and to explore its potential value in anti-Pseudomonas aeruginosa biofilm formation. Methods The antisense oligodeoxynucleotides (ODNs) which could bind to motA mRNA, a key gene of early biofilm formation in Pseudomonas aeruginosa PAO1, were screened by computer software and dot blot hybridization. The antisense oligodeoxynucleotides CCPs-PNAs were formed by cell membrane-bound peptide (CCP) (KFF) 3K, and Pseudomonas aeruginosa PAO1 was treated with different concentrations of CCPs, PNA and CCPs-PNA, respectively. Inhibition. Results PNA and CCPs-PNA inhibited the early formation of Pseudomonas aeruginosa PAO1 biofilm, and the inhibitory effect was also increased with the increase of the concentration of PNA and CCPs-PNA, but the inhibition of biofilm formation by CCPs-PNA was significantly better than that of PNA group. The inhibitory rates of 1,5,10 μmol / L (KFF) 3K-PNA on PAO1 biofilm formation were about 30%, 50% and 70%, respectively. While 1μmol / L PNA had no effect on PAO1 biofilm formation, the inhibitory rates of 5,10μmol / L PNA on PAO1 biofilm formation were about 3% and 10%, respectively. CCPs at all concentrations had no effect on bacterial biofilm formation. Compared with the control group, the exercise capacity of PAO1 after CCPs-PNA treatment decreased significantly, and the diffusion diameter ratio of PAO1-treated group and control group on exercise medium was about 3: 5. Conclusions The PNA against motA gene can inhibit the growth of Pseudomonas aeruginosa PA01 in the initial stage by inhibiting the movement ability of PAO1 and decreasing the adsorption of P01. KFV 3 K greatly enhances this effect. It is speculated that the motA gene of Pseudomonas aeruginosa or its encoded product may be a good target for biofilm formation against Pseudomonas aeruginosa.