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Title: 產黃纖維單胞桿菌幾丁質酶基因之選殖
Chitinase gene cloning from Cellulomonas flavigena NTOU 1
Authors: Pei-Wen Lin
Contributors: NTOU:Institute of Bioscience and Biotechnology
Keywords: 幾丁質酶;產黃纖維單胞桿菌
chitinase;Cellulomonas flavigena NTOU 1
Date: 2002
Issue Date: 2011-07-04
Abstract: 以含有蝦殼粉末為碳源的培養基,來誘導產黃纖維單胞桿菌(Cellulomonas flavigena NTOU 1)產生的幾丁質酶,經聚丙烯醯胺凝膠蛋白質電泳分離出有活性的幾丁質酶。將具活性的蛋白質轉漬到PVDF膜上,並定出其N端胺基酸序列及Q-TOF所定出部分片段的胺基酸序列。 參考已發表的其他菌種幾丁質酶胺基酸序列,取催化功能區相似度高的區域,及Q-TOF定出部分片段的胺基酸序列,設計了退化性引子。NTOU1-R和NTOU1-ASf進行聚合酶鏈反應(Polymerase Chain Reaction),可複製出約0.5 Kb的DNA片段,經核苷酸定序及電腦分析其胺基酸序列後,可轉譯成三個部份開放讀架(partial open reading frame),含有150,170及170個胺基酸。由此開放讀架DNA片段設計專一性引子,NTOU1-R和NTOU1-F2再進行聚合酶鏈反應,複製出約0.4 Kb的DNA片段,經選殖、核苷酸定序及電腦分析胺基酸序列後,可轉譯成四組150個胺基酸的部份開放讀架。將此部分開放讀架DNA片段選殖於表現載體pETBlue-2,做為日後進行南方點墨法分析的基因探針。 專一性引子NTOU1-F1或NTOU1-R1,及Random hexamer,所進行的Random Primed Gene Walking PCR去找出其基因的5’及3’端,但沒有成功。
The chitinase from Cellulomonas flavigena NTOU 1 was induced by growing the microorganism in a basal medium containing the chitin powder. The active chitinase was purified by polyacrylamide gel electrophoresis. The putative N-terminal amino acid of chitinase and its Q-TOF analysis are carried out using the purified protein transblotted to PVDF membrane. Degenerate primers were designed according to the published amino acid sequence of the enzyme highly conserved regions such as the catalytic domain from other bacterial chitinases and the partial amino acid sequence from this Q-TOF analysis. A DNA fragment of 0.5 Kb was amplified by PCR using the primer pairs of NTOU1-R and NTOU1-ASf. Three putative partial open reading frames encoding 150, 170, and 170 amino acids, respectively, were found from this 0.5 Kb DNA fragment from its nucleotide sequence analysis. Specific primers were then designed according to the putative partial open reading frame. A DNA fragment of 0.4 Kb was produced by PCR using the primer pairs of NTOU1-R and NTOU1-F2. Four putative partial open reading frames encoding 150 amino acids were available in this 0.4 Kb DNA. This putative partial 0.4 Kb gene fragment was cloned into the pETBlue-2 expression system and this fragment will be served as a gene probe in Southern blotting for its full-length chitinase gene cloning. There was no progress in Random Primed Gene Walking PCR using the primer pairs of NTOU1-F1 or NTOU1-R1 and random hexamer in order to search for the 5’- and 3’- ends of the chitinase gene.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0000000020
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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