靶向ftsZ基因反义肽肽核酸抑制耐甲氧西林的金黄色葡萄球菌的实验研究

发布时间：2018-01-29 12:42

本文关键词： 耐甲氧西林金黄色葡萄球菌 ftsZ 反义寡核苷酸斑点杂交肽核酸 ftsz 耐甲氧西林金黄色葡萄球菌　出处：《重庆医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：第一部分计算机辅助软件设计联合体外斑点杂交筛选靶向ftsZ基因反义寡核苷酸序列目的：采用计算机辅助软件设计联合体外斑点杂交筛选能与耐甲氧西林金黄色葡萄球菌ftsZ基因mRNA紧密结合的高效反义寡核苷酸序列。方法：利用计算机辅助软件(RNA Structure 5.0)对ftsZmRNA进行二级结构分析计算,并进行自由能计算,在膨胀环,发卡等不稳定的结构区域设计10条反义寡核苷酸序列,另设计一条与ftsZ基因上的一段碱基序列完全相同的寡核苷酸序列作为阴性对照,合成反义探针,和体外转录并且用地高辛标记的ftsZ mRNA进行斑点杂交,根据信号的强弱,筛选出高效的反义寡核苷酸序列。结果：体外斑点杂交实验结果显示,计算机软件设计的11条反义寡核苷酸序列中,其中有6条显示不同程度的杂交信号,1条正义序列未显示任何信号。结论：计算机辅助软件联合体外斑点杂交能够减少反义核酸设计的盲目性,为体外可靠、快速、高通量筛选高效反义寡核苷酸序列提供了一种有效的方法,并且为筛选所有基因高效反义序列搭建了一个平台。第三部分靶向ftsZ基因反义肽肽核酸体外抑制耐甲氧西林金黄色葡萄球菌生长目的：研究以fstZ基因为靶基因的反义肽核酸(peptide nucleic acid, PNA)对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA) fstZ基因表达的抑制作用和体外抗菌活性,探讨其作为新型抗菌药物制潜在应用价值。方法：在本实验研究中,根据第一部分筛选出的能与ftsZ基因高效紧密结合的反义寡核苷酸序列,合成肽核酸PNA1,同时,在ftsZ基因起始区域设计一条反义寡核苷酸序列,包括起始密码子AUG和SD序列,合成肽核酸PNA2,在PNA1的基础上设计一条有3个碱基错配的PNA1作为阴性对照,三条肽核酸分别与穿膜肽序列(RXR)4XB连接形成PPNA。分别用不同浓度的PPNA处理MRSA CY-11菌株,370c5%C02培养不同时间后测定细菌光密度值OD600并每隔两小时做平板菌落计数,观察其对细菌的生长抑制作用,采用逆转录PCR检测ftsZ基因mRNA表达水平观察其对靶基因表达的抑制作用。结果：PPNA1, PPNA2均具有明显的靶基因抑制作用和体外抗菌活性,并呈时间和浓度依赖性,其最低抑菌浓度分别为30μmol/L和40PPNA1在剂量为40 μmol/L具有杀菌活性,而具有3个错配碱基的Scr PPNA1对细菌生长无抑制作用,即使将其浓度增加到40μmol/L, RT-PCR结果表明反义肽核酸对ftsZ基因]mRNA表达水平的抑制呈浓度依赖性。结论：以ftsZ基因为靶点的PNA可在体外高效抑制ftsZ基因的表达和MRSA的生长,其反义抑制作用具有特异性,这项研究结果为针对MRSA的抗菌药物提供一种新的思路。
[Abstract]:Part one: computer aided software design for screening antisense oligonucleotide sequences targeting ftsZ gene by combined external dot blot. A highly efficient antisense oligonucleotide sequence which could bind to the ftsZ gene mRNA of methicillin-resistant Staphylococcus aureus was designed by computer aided software. Use of computer aided software (. RNA Structure 5.0) the secondary structure of ftsZmRNA was analyzed and calculated. The free energy was calculated and 10 antisense oligonucleotides were designed in unstable structural regions such as expansion rings and hairpins. In addition, an oligonucleotide sequence identical to a sequence of bases in ftsZ gene was designed as negative control to synthesize antisense probe. The antisense oligonucleotide sequence was screened by dot blot hybridization with ftsZ mRNA which was transcribed in vitro and labeled with digoxin. Results: the results of dot-blot hybridization in vitro showed that the antisense oligonucleotide sequence was highly effective. Of the 11 antisense oligonucleotide sequences designed by computer software, 6 showed hybridization signals of varying degrees. Conclusion: computer aided software combination with external dot hybridization can reduce the blindness of antisense nucleic acid design and is reliable and rapid in vitro. High throughput screening of high efficiency antisense oligonucleotide sequences provides an effective method. The aim of the third part was to inhibit the growth of methicillin-resistant Staphylococcus aureus in vitro by targeting the ftsZ gene antisense peptide peptide nucleic acid. Study on antisense Peptide Nucleic Acid with fstZ as Target Gene. Peptide nucleic acid. To methicillin-resistant Staphylococcus aureus. The inhibitory effect of fstZ gene expression and its antibacterial activity in vitro were investigated. Methods: in this study, the potential application value of MRSA as a new antimicrobial agent was discussed. According to the antisense oligonucleotide sequence of ftsZ gene which was screened out in the first part, the peptide nucleic acid PNA1 was synthesized. At the same time, the peptide nucleic acid PNA1 was synthesized. An antisense oligonucleotide sequence was designed in the initiation region of the ftsZ gene, including the initiation codon AUG and SD sequence, to synthesize the peptide nucleic acid PNA2. On the basis of PNA1, a PNA1 with three base mismatches was designed as a negative control. Three peptide nucleic acids were linked to the transmembrane peptide sequence RXRX4 XB to form PPNAs. MRSA CY-11 strains were treated with different concentrations of PPNA. The bacterial optical density (OD600) was measured after 370c52 culture for different time, and the colony count was made every two hours to observe its inhibitory effect on the growth of bacteria. The mRNA expression of ftsZ gene was detected by reverse transcription-polymerase chain reaction (PCR). PPNA2 has obvious target gene inhibitory effect and antibacterial activity in vitro, and it is time-and concentration-dependent. The minimum inhibitory concentration (MEC) was 30 渭 mol/L and 40PPNA1, respectively, with bactericidal activity at the dose of 40 渭 mol/L. However, Scr PPNA1 with three mismatched bases had no inhibitory effect on bacterial growth, even if its concentration was increased to 40 渭 mol/L. RT-PCR results showed that antisense peptide nucleic acid inhibited the expression of ftsZ gene] mRNA in a concentration-dependent manner. PNA targeting ftsZ could effectively inhibit the expression of ftsZ gene and the growth of MRSA in vitro. The antisense inhibitory effect is specific. This study provides a new idea for antimicrobial agents against MRSA.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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