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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 the antisense peptide sequence targeting lasR gene of Pseudomonas aeruginosa and investigate its effect on the biofilm formation ability of Pseudomonas aeruginosa. Methods Based on the lasR gene of Pseudomonas aeruginosa, four antisense oligonucleotide sequences were designed and the antisense oligonucleotide sequence with the best lasR gene was screened by dot blot hybridization to synthesize the peptide linked to the transmembrane peptide Peptide nucleic acid sequence. The different concentrations of peptide-peptide nucleic acid were respectively introduced into the Pseudomonas aeruginosa biofilm PAO1. The optical density [D (600)] of the bacteria at different time points was measured and the colonies were counted. Inhibition of growth. The formation of biofilm was observed by light microscopy and scanning electron microscopy. q PCR method to detect lasR mRNA expression. Results The results of dot blot hybridization showed that three of the four antisense oligonucleotides had hybridization signals, of which the first was the strongest signal and the antisense peptide was synthesized. Different concentrations of peptide - peptide nucleic acid showed different degrees of in vitro antibacterial activity against Pseudomonas aeruginosa, and with increasing concentration, antibacterial activity increased. Conclusions The antisense nucleotide sequence of lasR gene targeting P. aeruginosa was screened out by efficient screening of antisense nucleotide sequences. The antisense peptide nucleic acid of lasR gene of P. aeruginosa was able to inhibit the growth and biofilm formation of P. aeruginosa while inhibiting lasR mRNA expression.