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研究生:林良怡
研究生(外文):Liang-Yi Lin
論文名稱:表現及特徵化新穎之人類和斑馬魚17-beta羥基固醇去氫酶
論文名稱(外文):Expression and Characterization of A Novel 17-beta Hydroxysteroid Dehydrogenase of Human and Zebrafish
指導教授:劉德中劉德中引用關係
指導教授(外文):Te-Chung Liu
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:營養科學研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:102
中文關鍵詞:生物資訊資料庫17-beta羥基固醇去氫酶
外文關鍵詞:National Center for Biotechnology Information17-beta Hydroxysteroid Dehydrogenase
相關次數:
  • 被引用被引用:1
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  • 下載下載:10
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摘要
經由生物資訊資料庫(National Center for Biotechnology Information,NCBI)之搜尋和比對方式找尋到一組在斑馬魚(編號AI477544)及人類(編號NP_115679)皆有表現之新穎17-beta羥基固醇去氫酶(17-beta Hydroxysteroid Dehydrogenase,17β HSD)基因。利用3’ Rapid Amplification of cDNA Ends(3’RACE)聚合酶連鎖反應的方法,以0~7天之斑馬魚胚胎之基因庫為模板,成功地放大並定序其核酸序列。此斑馬魚之開放閱讀框架(Open Reading Frame)具有1469個鹼基對,推定之胺基酸為416個;人類之開放閱讀框架為3003個鹼基對,345個胺基酸。此新穎之17-beta HSD具有短鏈酒精去氫酶(adh_short)與膽固醇攜帶蛋白(SCP2)二個酵素之結合結構區,因此假定此酵素之生理功能及酵素反應相似於固醇類物質之氧化還原酶。利用pQE表達系統,在大腸桿菌(E coli.)中表現及純化該斑馬魚及人類之新穎17-beta羥基固醇去氫酶(17β HSD)之蛋白質後,利用酵素動力學分析(enzyme kinetic assay)方法進行酵素反應之分析測試。結果發現此酵素以NAD+/NADP+為輔酶,Estriol和5-Androstene 3β,16α,17β -triol為受質時,可推動受質的氧化作用,因此該酵素可能是16α,17β dihydroxysteroid dehydrogenase。
ABSTRACT
In this study, by using BLAST (Basic Local Alignment Search Tool), a novel 17-beta Hydroxysteroid Dehydrogenase of zebra fish(accession number AI477544) and human(accession number NP_115679) were found from NCBI (National Center for Biotechnology Information) nucleotide database. The novel 17-beta Hydroxysteroid Dehydrogenase cDNA of zebra fish was successfully amplified from the 0-7 days old zebra fish embryo cDNA library by using the combination of PCR and 3’rapid-amplification of cDNA ends (3’RACE) methods. The novel zebra fish cDNA had a 1469 bp open reading frame and 416 deduced amino acid. The novel human cDNA had a 3303bp open reading frame and 345 amino acid. Both enzyme had two domain, i.e. short chain alcohol dehydrogenase(adh_short) and steroid carried protein(SCP2)domain. The initial velocity analysis of both enzyme showed that two substrates, Estriol and 5-Androstene 3β,16α,17β-triol may participate in the catalytic reaction of the novel 17-beta Hydroxysteroid Dehydrogenase.
中文摘要 I
英文摘要 III
目錄 IV
圖表次 VI
附錄文次 VII
中英文對照表 VIII
壹、緒論 1
一 基因體學與生物資訊學的興起 1
二 功能性基因體學與蛋白質體學之未來發展 3
三 本研究動機與方向 7
四 本研究之概況與研究目的 10
貳、材料與方法 14
一 NCBI生物資訊網路搜尋 14
(一) Entrez 14
(二) BLAST 14
(三) 生物資訊應用軟體之分析/處理 16
(四) 其他工具網站之應用 17
二 材料與實驗方法 19
(一) 材料 19
(二) 延長及放大原始Z-HSD完整序列 20
(三) 建構H-HSD與Z-HSD全長核酸序列 25
(四) 建構蛋白質表達質體 28
(五) 蛋白質大量表達、純化與定量分析 30
參、 結果 36
一 生物資訊資料庫(NCBI)搜尋 36
二 斑馬魚(Z-HSD)全長序列之選殖與定序 38
三 人類(H-HSD)全長序列之選殖與定序 39
四 人類與斑馬魚之(HSD)蛋白質表達質體之建構 39
五 新穎蛋白質之表達與純化 40
六 酵素反應分析 41
肆、 討論 43
伍、 參考文獻 47
陸、 圖表 53
柒、 附錄 78
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