结核杆菌DNA回旋44酶B亚基的克隆表达及其抑制剂的分离纯化研究

发布时间：2018-03-09 13:31

本文选题：DNA回旋酶　切入点：结核　出处：《中国协和医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： DNA回旋酶是细菌所特有的一种拓扑异构酶,该酶由两个A亚基和两个B亚基聚合而成。A、B亚基分别有各自的功能区,共同决定该酶的生物学功能。它能够利用ATP的能量将共价闭合环状DNA(cccDNA)转变为负超螺旋DNA,从而维持细菌体内正常的超螺旋水平。筛选结核杆菌回旋酶抑制剂有可能获得新型抗结核药物。本课题组应用细胞水平的以DNA回旋酶B亚基为靶点的抗结核药物筛选模型,筛选得到了25个阳性菌株,本研究对这些菌株进行了再筛选,对活性稳定的阳性菌株I03A-09005和I03A-00463进行深入研究。菌株I03A-09005的发酵液经大孔树脂柱层析,硅胶柱,HPLC分离得到了单一组分的活性化合物9005B,该化合物对H37Rv的MIC为64～128 ug/ml,并经过质谱、碳谱、氢谱等进行结构确证研究,鉴定9005B为放线菌素X2。菌株I03A-00463的发酵液经大孔树脂柱层析,ODS柱层析,得到纯度约为28.15%的阳性化合物03-0463。同时,采用分子生物学方法构建了含有结核杆菌H37Rv gyrB基因的表达质粒pET-19b/gyrB,转化至BL21(λDE3)pLysS中,利用原核表达系统成功克隆表达了结核杆菌H37Rv的DNA回旋酶B亚基,为建立分子水平得以DNA回旋酶B亚基为靶点的抗结核药物筛选模型奠定了基础。
[Abstract]:DNA gyrase is a topoisomerase specific bacteria, the enzyme is composed of two A subunits and two B subunit polymerization of.A and B subunits respectively have their respective functional areas, jointly determine the biological function of the enzyme. It can use the energy of ATP will be covalently closed circular DNA (cccDNA) change negative supercoiled DNA, so as to maintain the normal level of supercoiling in bacteria. Screening Mycobacterium tuberculosis gyrase inhibitors may obtain new anti tuberculosis drugs.
The level of the research group using cell with DNA gyrase B subunit as a screening model for drug screening, we obtained 25 positive strains, this study conducted a re screening of these strains, in-depth study of the active and stable positive strains I03A-09005 and I03A-00463. The fermentation broth of strain I03A-09005 by macroporous resin column chromatography, silica gel column, HPLC isolated from the single component of the active compound 9005B, the compound of H37Rv MIC was 64~128 ug/ml, and after the mass spectrum of carbon, hydrogen spectrum of structure identification, identification of 9005B fermentation liquid of actinomycin X2. strain I03A-00463 by macroporous resin column chromatography, ODS column chromatography, the purity of about 28.15% positive compounds 03-0463. and plasmid pET-19b/gyrB containing H37Rv of Mycobacterium tuberculosis gyrB gene using molecular biology methods, conversion to BL21 (2 DE3) pLysS in use The prokaryotic expression system successfully cloned and expressed the DNA cyclogyrase B subunit of Mycobacterium tuberculosis H37Rv, which laid the foundation for establishing the screening model of anti tuberculosis drugs targeting at the molecular level and the DNA cyclogyrase B subunit.

【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378

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