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dc.contributor.author胡青穎en_US
dc.contributor.authorChing-Ying Hwuen_US
dc.contributor.author吳東昆en_US
dc.contributor.authorTung-Kung Wuen_US
dc.date.accessioned2014-12-12T02:29:54Z-
dc.date.available2014-12-12T02:29:54Z-
dc.date.issued2002en_US
dc.identifier.urihttp://140.113.39.130/cdrfb3/record/nctu/#NT910111002en_US
dc.identifier.urihttp://hdl.handle.net/11536/69830-
dc.description.abstractβ-lactam類抗生素被廣泛的使用在治療與細菌感染有關之疾病上,但由於某些菌種產生具抗藥性的β-lactamase,而使得此類抗生素使用被限制不少。Clavulanic acid為一含氧的雙環β-lactam類抗生素,而其特殊立體結構使得clavulanic acid成為β-lactamase酵素之有效抑制劑。其抗菌活性來自於其β-lactam的四圓環及特殊第三個及第五個碳上之官能基的立體結構,不同於其他β-lactam類抗生素,因此拿來與其它他β-lactam類抗生素合併使用,應用在會產生β-lactamase酵素之菌株上。例如clavulanic acid與 amoxicillin混合的藥品Augmentin®;或與ticarcillin混合之藥品為Timentin®都已上市銷售,因此對於clavulanic acid這個天然產物,大量生產對於工業製藥上具有極高的經濟價值。 為了了解如何調節clavulanic acid的產生,我們針對位於此抗生素生合成基因組中,可能扮演調控因子之claR基因,其所轉譯之蛋白質ClaR作相關研究。ClaR蛋白質之分子量約為47 kDa,經序列比對後發現與LysR家族這類轉錄調控因子有很高相似性,帶有可與DNA結合特性之Helix-Turn-Helix (HTH) 高度保留序列。因此先把ClaR蛋白質與麥芽醣結合蛋白 (MBP)產生融合蛋白,利用通過親合性管柱 (amylose column)層析純化後,得到分子量約90 kDa之融合蛋白質。利用指數放大的系統性配位基演化 (SELEX)技術的原理,從一個含有隨機核酸序列的組合中,針對蛋白質ClaR找出具有專一性的一段核酸序列,使用親合性固相結合分析及電泳遲滯分析法偵測核酸與蛋白質之複合體,利用聚合酵素連鎖反應、分生選殖技術、核酸之定序等方式來尋找此具專一性之核酸並比對這些核酸序列與clavulanic acid生合成基因之關聯性。 因此我們針對MBP-ClaR融合蛋白,經過SELEX技術篩選並定序之,目前得到了13個核酸aptamer。並經由生物資訊來分析之,發現這序列不盡相同的13個核酸aptamer當中的11個序列,於不同的位置上皆可找到一段五個鹼基帶有GGGGG, GCGGG, GGCGG or GGGCG的GC含量高之核酸組合序列。zh_TW
dc.description.abstractβ-lactam antibiotics are widely used to treat bacterial infection. The inactivation of β-lactam antibiotics by β-lactamase-producing organisms limits the effectiveness of these compounds in treating infection, however. Clavulanic acid (CA), an oxygen-containing bicyclic β-lactam antibiotic produced by Streptomyces clavuligerus, is a potent β-lactamase inhibitor. The antimicrobial efficacy of CA is derived from its β-lactam ring and specific 3R, 5R stereochemistry different from other β-lactam antibiotics. Today, combinations of amoxicillin plus CA (Augmentin® ) and of ticarcillin plus CA (Timentin®) have been widely successful in the treatment of infection caused by β-lactamase-producing bacteria resistant to amoxicillin and ticarcillin. In order to understand the regulation of clavulanic acid production, we worked on the identification of DNA binding site of claR gene, a putative β-lactam regulatory gene located within the clavulanic acid biosynthetic gene cluster. ClaR gene encodes a polypeptide of 47 kDa with helix-turn-helix motifs homologous to DNA-binding proteins of the LysR family. The ClaR protein was over-expressed as a fusion to the maltose-binding protein (MBP) and purified by amylose affinity column as a ~90 kDa MBP-ClaR fusion protein. A SELEX technology (Systematic Evolution of Ligand by EXponential enrichment) was applied to isolate aptamers from the pool that can bind to the ClaR protein with high affinity and specificity. The SELEX procedures include iterative selection and amplification of DNA ligands that can bind ClaR protein. Both matrix binding assay and gel mobility shift assay were used to detect DNA-protein complex. The specific oligonucleotide sequences obtained from polymerase chain reaction, molecular cloning, DNA sequencing for their relationships relative to the clavulanic acid biosynthetic gene cluster. Following the selection of DNA aptamer to MBP-ClaR fusion protein, 13 DNA aptamer clones were sequenced and subjected to bioinformatic analysis. A pentamer GGGGG, GCGGG, GGCGG or GGGCG sequence was found at a variable location with the randomized region in 11 out of 13 clones.en_US
dc.language.isozh_TWen_US
dc.subject指數放大的系統性配位基演化技術zh_TW
dc.subjectClavulanic aciden_US
dc.subjectSELEXen_US
dc.subjectClaRen_US
dc.title利用指數放大的系統性配位基演化技術篩選辨識clavulanic acid生合成途徑中轉錄因子ClaR之DNA序列zh_TW
dc.titleIdentification of a DNA aptamer for the transcriptional factor ClaR of clavulanic acid in Streptomyces clavuligerus. Application of systematic evolution of ligands by exponential enrichment. (SELEX)en_US
dc.typeThesisen_US
dc.contributor.department生物科技學系zh_TW
Appears in Collections:Thesis