靶向motA反义肽肽核酸体外抑制铜绿假单胞菌PAO1生物膜形成的研究

发布时间：2018-07-10 05:43

本文选题：铜绿假单胞菌 + 斑点杂交　；参考：《重庆医科大学》2011年硕士论文

【摘要】：第一部分计算机辅助设计联合斑点杂交筛选铜绿假单胞菌PAO1motA基因反义寡核苷酸序列目的:采用全基因寻靶技术(full length gene targeting, FLGT)筛选能与铜绿假单胞菌PAO1motA基因的mRNA结合紧密的反义寡核苷酸序列。方法:采用计算机软件(Mfold和RNA Structure 4.6)模拟铜绿假单胞菌PAO1motA基因mRNA的二级结构,根据最小自由能原理设计出6条反义寡核苷酸探针序列,另设两条阴性对照:一条为随机设计的阴性对照序列,另一条为与motA基因上的一段碱基完全一样的序列。克隆motA基因并进行体外转录,同时用地高辛标记motA mRNA,以斑点杂交方法筛选出与motA基因mRNA结合紧密、杂交信号较强的反义寡核苷酸序列。结果:PCR扩增出motA基因的全长(852bp),斑点杂交显示6条反义寡核苷酸中的4条显示出杂交信号。结论:成功筛选到了能与motA mRNA牢固结合的反义序列,为进一步研究以motA基因为靶的反义技术抑制生物膜形成打下基础。第二部分肽核酸体外抑制铜绿假单胞菌PAO1生物膜形成的研究目的:研究肽核酸(peptide nucleic acid,PNA)对铜绿假单胞菌PAO1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:根据第一部分筛选出的的反义寡核苷酸的序列合成肽核酸(PNA)并连接穿膜肽(cell penetrating 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,同时检测其对motA基因表达的抑制作用以及对PAO1运动能力的影响。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生物膜的形成基本上没有影响,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基本上没有影响。PNA和CCPs-PNA对motA基因的表达均有抑制作用,但CCPs-PNA的抑制明显优于PNA组。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具有抑制作用,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。
[Abstract]:Part one screening of antisense oligonucleotide sequence of PAO1motA gene of Pseudomonas aeruginosa by computer-aided design combined with dot blot objective: to screen PAO1motA gene of Pseudomonas aeruginosa by (full length gene targeting, FLGT Its mRNA binds closely to the antisense oligonucleotide sequence. Methods: using computer software (Mfold and RNA structure 4.6) to simulate the secondary structure of PAO1motA gene mRNA in Pseudomonas aeruginosa, six antisense oligonucleotide probes were designed according to the principle of minimum free energy. Another two negative controls: one was a randomly designed negative control sequence and the other was a sequence identical to a sequence of bases on the motA gene. MotA gene was cloned and transcribed in vitro. At the same time, motA mRNAs were labeled with digoxigenin. The antisense oligonucleotide sequences with strong hybridization signal were screened by dot blot hybridization. Results the full length (852bp) of motA gene was amplified by motA and 4 of 6 antisense oligonucleotides were detected by dot blot hybridization. Conclusion: the antisense sequence which can bind to motA mRNA has been successfully screened, which lays a foundation for further research on the inhibition of biofilm formation by the antisense technique based on motA. The second part of the study on the inhibition of PAO1 biofilm formation of Pseudomonas aeruginosa in vitro by peptide nucleic acid (PNA) objective: to study the inhibitory effect of PNA on PAO1 biofilm formation of Pseudomonas aeruginosa in vitro. To explore the application value of its potential anti-Pseudomonas aeruginosa biofilm formation. Methods: according to the sequence of antisense oligodeoxynucleotides selected in the first part, synthetic peptide nucleic acid (PNA) was synthesized and ligated with transmembrane peptide (cell penetrating peptide, CCPs (KFF) 3K to form CCPs-PNA.The PAO1 of Pseudomonas aeruginosa was treated with different concentrations of CCPs, PNA and CCPs-PNA, respectively. The inhibition of biofilm formation was observed by microscope. At the same time, the inhibition of motA gene expression and its effect on PAO1 motor ability were detected. Results both of them inhibited the early formation of PAO1 biofilm of Pseudomonas aeruginosa, and with the increase of concentration, the inhibitory effects of PNA and CCPs-PNA were also increased. However, the inhibition of biofilm formation by CCPs-PNA was significantly higher than that by (KFF) 3K-PNA (KFF) 3K-PNA (10 渭 mol / L) in PNA group. The inhibitory rates of CCPs-PNA on PAO1 biofilm formation were about 3050g / 70g / L, respectively. However, 1 渭 mol / L PNA had no effect on the formation of PAO1 biofilm, but the inhibition rates of 10 渭 mol / L PNA on PAO1 biofilm formation were about 3% and 10%, respectively. Different concentrations of CCPs had no effect on the formation of bacterial biofilm. PNA and CCPs-PNA could inhibit the expression of motA gene, but the inhibition of CCPs-PNA was better than that of PNA group. Compared with the control group, the motor ability of PAO1 treated with CCPs-PNA was significantly decreased. The diffusive diameter ratio of PAO1 treatment group to control group was about 3: 5. Conclusion: PNAs targeting motA gene can inhibit the biofilm formation of Pseudomonas aeruginosa PA01 at the initial stage by inhibiting the motor ability of PAO1 and reducing the adsorption, which is greatly enhanced by the transmembrane peptide (KFF) 3K. It is speculated that the motA gene or its encoding product of Pseudomonas aeruginosa may be a good target for the biofilm formation of Pseudomonas aeruginosa.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R440;R378.991

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