選殖與表達人類及斑馬魚之雙氫雙羥基去氫

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Title:	選殖與表達人類及斑馬魚之雙氫雙羥基去氫 Cloning and Expression a Dimeric Dihydrodiol Dehydrogenase of Human and Zebrafish
Authors:	馬千茹 Chien-Ju Ma
Contributors:	中山醫學大學：營養學研究所;劉德中
Keywords:	國家生物資訊中心;雙氫雙羥基去氫;最初反應速率 National Center for Biotechnology Information;Dimeric Dihydrodiol Dehydrogenase;initial velocity kinetic study
Date:	2005/07/06
Issue Date:	2010-03-17T07:01:14Z (UTC)
Abstract:	利用生物資訊資料庫NCBI（National Center for Biotechnology Information）中的搜尋比對工具BLAST（Basic Local Alignment Search Tool），搜尋比對到一組相對應的斑馬魚（編號BI326319）與人類（編號NM_014475.2/NP_055290）雙氫雙羥基去氫。此斑馬魚雙氫雙羥基去氫cDNA之開放閱讀框架（Open Reading Frame）含有1002個鹼基對，可轉譯成299個胺基酸，估計分子量36.63kDa；而人類已完全被放大出來，人類雙氫雙羥基去氫cDNA之開放閱讀框架含有1005個鹼基對，可轉譯成334個胺基酸，估計分子量36.74kDa。藉由pQE表達質體系統，將斑馬魚與人類雙氫雙羥基去氫表達與純化出來，再以12％ SDS-PAGE分析此重組蛋白質，結果顯示斑馬魚和人類蛋白質分子量如預期所估計。此酵素經北方墨點分析(Northern Blotting)，發現在大鼠的腦、心、肺、脾、腎、腸、肌肉、睪丸皆有表現。利用酵素動力學分析（Enzyme kinetic assay）測試酵素反應，結果發現此斑馬魚以NAD+為輔，以Maltose、D-Glucose、D-Mannose、D-Xylose、D-Fructose、D-Arabinose、 D-Ribose及D-Galactose為受質，其比活性分別為186、170、146、140、136、54、41unit/mg。因此，推斷此酵素可能為新穎之雙氫雙羥基去氫。 In this study, the srarch tool BLAST(Basic Local Alignment Search Tool), based on NCBI（National Center for Biotechnology Information）was used against nucleotide and protein database. The analysis found a Dimeric Dihydrodiol Dehydrogenase（dimeric DD）of zebrafish（NCBI accession number BI326319）and human（NCBI accession number NM_014475.2/NP_055290）.The dimeric DD cDNA of zebrafish and human were successfully amplified with a continuous open reading frame of 1002 and 1005 bps encoding a protein of 299 and 334 amino acid with a calculated molecular mass of 36.63 and 36.74 kDa, respectively. Zebrafish and human dimeric DD were cloned and expressed with a 6-Histidine tag for specific purification. The purified recombinant protein have mobility of 36.63 kDa and 36.74 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Northern Blotting analysis showed that dimeric DD is expressed in the rat tissues, such as brain, heart, lung, liver, spleen, intestine, kidney, muscle, and testis. Following expression, purification, and the kinetic studies, using Maltose、D-Glucose、D-Mannose、D-Xylose、D-Fructose、D-Arabinose、D-Ribose、D-Galactose and NAD+ as substrates, the specific activities of Ni2+ column purified recombinant zebrafish dimeric DD were 186、170、146、140、136、54、41 unit/mg, respectively. Therefore, this enzyme may belong to the novel Dimeric Dihydrodiol Dehydrogenase, which uses carbohydrate as substrates.
URI:	http://140.128.138.153:8080/handle/310902500/788
Appears in Collections:	[School of Nutritional Science] Electronic Theses and Dissertation

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