本文關鍵詞： 耐甲氧西林金黃色葡萄球菌 ftsZ 反義寡核苷酸 斑點雜交 肽核酸 ftsz 耐甲氧西林金黃色葡萄球菌　出處：《重慶醫(yī)科大學》2015年碩士論文　論文類型：學位論文

【摘要】：第一部分計算機輔助軟件設計聯(lián)合體外斑點雜交篩選靶向ftsZ基因反義寡核苷酸序列目的：采用計算機輔助軟件設計聯(lián)合體外斑點雜交篩選能與耐甲氧西林金黃色葡萄球菌ftsZ基因mRNA緊密結合的高效反義寡核苷酸序列。方法：利用計算機輔助軟件(RNA Structure 5.0)對ftsZmRNA進行二級結構分析計算,并進行自由能計算,在膨脹環(huán),發(fā)卡等不穩(wěn)定的結構區(qū)域設計10條反義寡核苷酸序列,另設計一條與ftsZ基因上的一段堿基序列完全相同的寡核苷酸序列作為陰性對照,合成反義探針,和體外轉錄并且用地高辛標記的ftsZ mRNA進行斑點雜交,根據(jù)信號的強弱,篩選出高效的反義寡核苷酸序列。結果：體外斑點雜交實驗結果顯示,計算機軟件設計的11條反義寡核苷酸序列中,其中有6條顯示不同程度的雜交信號,1條正義序列未顯示任何信號。結論：計算機輔助軟件聯(lián)合體外斑點雜交能夠減少反義核酸設計的盲目性,為體外可靠、快速、高通量篩選高效反義寡核苷酸序列提供了一種有效的方法,并且為篩選所有基因高效反義序列搭建了一個平臺。第三部分靶向ftsZ基因反義肽肽核酸體外抑制耐甲氧西林金黃色葡萄球菌生長目的：研究以fstZ基因為靶基因的反義肽核酸(peptide nucleic acid, PNA)對耐甲氧西林金黃色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA) fstZ基因表達的抑制作用和體外抗菌活性,探討其作為新型抗菌藥物制潛在應用價值。方法：在本實驗研究中,根據(jù)第一部分篩選出的能與ftsZ基因高效緊密結合的反義寡核苷酸序列,合成肽核酸PNA1,同時,在ftsZ基因起始區(qū)域設計一條反義寡核苷酸序列,包括起始密碼子AUG和SD序列,合成肽核酸PNA2,在PNA1的基礎上設計一條有3個堿基錯配的PNA1作為陰性對照,三條肽核酸分別與穿膜肽序列(RXR)4XB連接形成PPNA。分別用不同濃度的PPNA處理MRSA CY-11菌株,370c5%C02培養(yǎng)不同時間后測定細菌光密度值OD600并每隔兩小時做平板菌落計數(shù),觀察其對細菌的生長抑制作用,采用逆轉錄PCR檢測ftsZ基因mRNA表達水平觀察其對靶基因表達的抑制作用。結果：PPNA1, PPNA2均具有明顯的靶基因抑制作用和體外抗菌活性,并呈時間和濃度依賴性,其最低抑菌濃度分別為30μmol/L和40PPNA1在劑量為40 μmol/L具有殺菌活性,而具有3個錯配堿基的Scr PPNA1對細菌生長無抑制作用,即使將其濃度增加到40μmol/L, RT-PCR結果表明反義肽核酸對ftsZ基因]mRNA表達水平的抑制呈濃度依賴性。結論：以ftsZ基因為靶點的PNA可在體外高效抑制ftsZ基因的表達和MRSA的生長,其反義抑制作用具有特異性,這項研究結果為針對MRSA的抗菌藥物提供一種新的思路。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R446.5

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本文編號：1473444