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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 分子生物學研究所 > 依資料類型分類 > 碩博士論文 >  Acinetobacter baumannii溶裂型噬菌體phiAbA53之分離與分析以及Xanthomonas oryzae pv.oryzae噬菌體phiXo411 ORF21之表現探討

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/92954

標題: Acinetobacter baumannii溶裂型噬菌體phiAbA53之分離與分析以及Xanthomonas oryzae pv.oryzae噬菌體phiXo411 ORF21之表現探討
Isolation and characterization of phiAbA53, a lytic phage infecting Acinetobacter baumannii, and expression of ORF21 of Xanthomonas oryzae pv. oryzae phage phiXo411
作者: 富勇傑
Fu, Yung-Chieh
Contributors: 翁淑芬
Shu-Fen Weng
中興大學
關鍵字: 噬菌體;鮑氏不動桿菌;全抗藥性;黃原菌;溶菌酶;電顯;脈衝式電泳
phage;acinetobacter baumannii;pan-drug resistance;xanthomonas;lysozyme;TEM;pulse-field electrophoresis;zymogram assay;pichia pastoris
日期: 2007
Issue Date: 2012-08-31 12:09:33 (UTC+8)
Publisher: 分子生物學研究所
摘要: 第一部份
由病人的痰液檢體中篩選到一株可專一感染 Acinetobacter baumannii 的溶裂型噬菌體,命名為 phiAbA53。以電子顯微鏡觀察,此噬菌體具有一個六角型的頭部,直徑約為 60 nm;以及可收縮的尾部,長度約為 14 x 143 nm。根據外觀型態,此噬菌體歸屬於 Myoviridae 的 A1 type。經限制酶切割圖譜以及脈衝式電泳分析,估算此噬菌體之基因體大小約為 95 kb。以 SDS-PAGE 分析 phiAbA53 的外套蛋白質組成,至少包含 20 條蛋白質條帶。根據單步生長曲線的結果,phiAbA53 的潛伏期約為 10 分鐘;隱晦期僅 5 分鐘,而世代時間約為 25 分鐘。感染 5 分鐘後即有 95% 的噬菌體顆粒吸附至宿主細胞上,每個被感染的宿主細胞約可釋放 100 個噬菌體子代顆粒。經構築基因庫與次選殖等方式將 phiAbA53 的部分DNA片段構築於 pOK12 或 yT&A 載體上,然後進行定序。目前已定序的序列約 20 kb。根據定序結果,以一 2.1 kb 包含噬菌體外殼蛋白質基因之片段為探針,進行噬菌體外殼蛋白質基因叢集區域之找尋,經由引子步查法 (primer walking) 的方式得到噬菌體外殼蛋白質基因片段及其上、下游序列共 10,290 bp。,經電腦分析比對,推測有 9 個 ORFs 存在。
第二部份
噬菌體 phiXo411 是一株專一感染 Xanthomonas oryzae pv. oryzae 之溶裂型噬菌體。其 orf21 基因產物的分子量大小約 16 kD,pI 5.3,推測為噬菌體的外殼蛋白質,具有peptidoglycan hydrolase 的性質。本實驗之目的為探討 ORF21 之功能與特性,以瞭解噬菌體 phiXo411 吸附至宿主表面後,ORF21 是否能水解細胞壁以協助 DNA 注入宿主細胞。本論文探討中,以純化的 ORF21 蛋白質為抗原,免疫兔子,製備具專一性之抗體。利用西方墨點法證實 ORF21 存在於 phiXo411 噬菌體外殼蛋白質中。上述抗體,亦可偵測到與噬菌體 phiXo411 ORF21 胺基酸序列達 57% 相似度的噬菌體 phiL7 外殼蛋白質 ORF19。此外,利用金粒子免疫電顯法測試可知,ORF21 位於 phiXo411 噬菌體的基板上。將包含 orf21 之 DNA 序列選殖於不同的表現載體中,進行蛋白質表現。在 E. coli 的蛋白質表現系統中無法得到具有活性的 ORF21。將酵母菌 P. pastoris KM71PAOX1::ORF21 胞外培養液中純化獲得之 sORF21His 蛋白質,點滴於含有 Xo21 的雙層培養基上可觀察到溶菌圈形成,顯示 ORF21 具有溶菌能力。在 Zymogram assay 中,將失活的 ORF21 蛋白質以適當緩衝液進行活性回復後,可分解 SDS-PAGE 電泳膠片中的 Xo21 peptidoglycan,上述結果可證實 ORF21 具有 peptidoglycan hydrolase 活性。
Part Ⅰ
A lytic bacteriophage infecting Acinetobacter baumannii specifically was isolated from sputum of the clinical patient. Electron microscopy revealed that the phage had an isomeric head (60 nm in diameter), a long contractile tail (143 nm in length and 14 nm in wide), and a complex baseplate with tail fibers. The phage belongs to Bradly A1 of Myoviridae family. A physical restriction digestion map and pulsed-field electrophoresis showed that the genome size was about 95 kb. SDS-PAGE profiles indicated that phage phiAbA53 contained 20 structural proteins at least. In addition, one step growth kinetic of the phage showed that the latent and eclipse periods were 10 and 5 min, and generation time was 25 min, respectively, with burst size of 100 PFU per infected cell. 90% of phage particles were absorbed to host cells 5 min after infection. Genomic library of the phiAbA53 was constructed on pOK12 or yT&A vector, and appropriately 20 kb of phiAbA53 genomic DNA was sequenced. According to the sequencing results, a 2.1 kb DNA fragment, containing partial minor capsid protein gene, was used as a probe to search for the capsid protein gene cluster of phage phiAbA53. And 10,290 bp, including the minor capsid protein gene and its upstream and downstream sequence, was obtained via primer walking. Sequence analysis results revealed 9 putative ORFs.
Part Ⅱ
phiXo411 is a lytic bacteriophage infecting Xanthomonas oryzae pv. oryzae. Sequence analysis results predicted that the gene product of orf21 is phage capsid protein, 16 kD in size, with an enzyme activity of peptidoglycan hydrolase. In this study, the function and characteristic of ORF21 were investigated. Using the purified ORF21His as an antigen to immunize New Island rabbit, the anti-ORF21 antibody was obtained. The result of Western blotting revealed the existence of ORF21 on the capsid of phiXo411. phiL7 ORF19, a homolog of phiXo411 ORF21 with 57% identity, was able to be detected by the same antibody after Western blotting. In addition, based on the study of immuno-gold linked TEM, the location of ORF21 was identified at the baseplate of phiXo411. The PCR product carrying the phiXo411 orf21 sequence was cloned in various vector systems, including pET30-a. pET32-a, pGEX-4T-1, and pPICB, to overexpress ORF21 protein. Although no enzymatic activity was detected in E. coli expression system, the soluble form of sORF21His was harvested and purified from the extracellular fraction of yeast transformant. A clear zone was observed after applying sORF21His on the double layer plate using Xo21 as indicator host. In addition, it was formed that denatured ORF21 could be renatured in the renaturing buffer and hydrolyzed the peptidoglycan included in the SDS-PAGE gel. These results demonstrated the peptidoglycan hydrolase activity on phiXo411 ORF21.
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