中大機構典藏-NCU Institutional Repository-提供博碩士論文、考古題、期刊論文、研究計畫等下載:Item 987654321/6216
English  |  正體中文  |  简体中文  |  全文笔数/总笔数 : 70585/70585 (100%)
造访人次 : 23155684      在线人数 : 462
RC Version 7.0 © Powered By DSPACE, MIT. Enhanced by NTU Library IR team.
搜寻范围 查询小技巧:
  • 您可在西文检索词汇前后加上"双引号",以获取较精准的检索结果
  • 若欲以作者姓名搜寻,建议至进阶搜寻限定作者字段,可获得较完整数据
  • 进阶搜寻


    jsp.display-item.identifier=請使用永久網址來引用或連結此文件: http://ir.lib.ncu.edu.tw/handle/987654321/6216


    题名: 絲瓜簇葉病植物菌質體胸;Expression and characterization of the thymidylate kinase from the phytoplasma associated with the loofah witches' broom
    作者: 江施幸;Shih-Hsing Chiang
    贡献者: 生命科學研究所
    关键词: 絲瓜簇葉病植物菌質體;;tmk;GST-TMK fusion protein;Km;Vmax;PK/LDH coupling reaction;phytoplasma
    日期: 2000-07-18
    上传时间: 2009-09-22 10:15:49 (UTC+8)
    出版者: 國立中央大學圖書館
    摘要: 本實驗為研究絲瓜簇葉病植物菌質體tmk 基因段。以引子tmk-1與 tmk-2,利用聚合?連鎖反應(PCR) 擴增絲瓜簇葉病植物菌質體tmk 基因段,再利用EcoR1 與 BamH1 限制?切位將此基因段構築至pGEX-2T 蛋白質表現載體之中。將此重組的載體表現在大腸桿菌XL1-blue寄主細胞中大量表現並且在IPTG誘導之下生合成由GST與胸?酸激?(TMK)組成之融合蛋白。此蛋白質為分子量大約50 kD, 在應用親和性Glutathione Sepharose 4B管柱萃取純化以及thrombin酵解之後便得到24 kD左右的胸?酸激?蛋白質。   此胸?酸激?催化磷酸根基團的轉移從ATP轉給dTMP。連續的分光儀分析加上運用丙酮酸磷酸?與乳酸去氫?做偶合反應,用來分析胸?酸激?酵素活性反應以及酵素動力學研究。在酵素活性反應中還原態的NADH被氧化並且在波長340 nm 吸光值發生變化,我們便可以觀察到胸?酸激?的活化催化作用。其反應pH 值約為pH 7 而鎂離子(Mg++)濃度為1.5 ∼ 2.0 mM 而最佳溫度為30 ℃。酵素胸?酸激?的km 與Vmax 值為 0.058 mM 和 1.967 μmol/min 。 The tmk gene fragments were amplified on the DNA of phytoplasma associated with loofah witches' broom by polymerase chain reaction (PCR) using the two primers, tmk-1 and tmk-2, and constructed into the pGEX-2T expression vector at EcoR1 and BamH1 sites. The recombinant vector was expressed in E. coli XL1-blue and a fusion protein composed of glutathione S-transferase (GST) and thymidylate kinase (TMK) was produced in the presence of IPTG. The protein had a molecular weight of about 50 kD. After purified by Glutathione Sepharose 4B column and treated by thrombin, a TMK protein of about 24 kD was obtained. The enzyme TMK catalyzes the transfer of phosphate group from ATP to thymidine monophosphate. A continuous spectrophotometric assay using the pyruvate kinase (PK) and lactate dehydrogenase (LDH) coupling system was employed for the TMK activity assay. The reduced-form NADH was oxidized in the reaction and the absobance at 340 nm was measured. The optimum pH for the reaction was about 7 and Mg++ concentration was 1.5 ∼ 2.0 mM at 30 ℃. The km and Vmax of enzyme TMK was 0.058 mM and 1.967 μmol/min, respectively.
    显示于类别:[生命科學研究所 ] 博碩士論文

    文件中的档案:

    档案 大小格式浏览次数


    在NCUIR中所有的数据项都受到原著作权保护.

    社群 sharing

    ::: Copyright National Central University. | 國立中央大學圖書館版權所有 | 收藏本站 | 設為首頁 | 最佳瀏覽畫面: 1024*768 | 建站日期:8-24-2009 :::
    DSpace Software Copyright © 2002-2004  MIT &  Hewlett-Packard  /   Enhanced by   NTU Library IR team Copyright ©   - 回馈  - 隱私權政策聲明