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The Research on the Search for Biologically Active Natural Products Utilizing Sterol Biosynthetic Enzymes as the Target

以甾醇生物合成酶为目标寻找生物活性天然产物的研究

基本信息

批准号：
07557372
负责人：
EBIZUKA Yutaka
金额：
$ 0.9万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557372/
关键词：
Biosynthesis of Sterols Lanosterol Synthase Gene Cloning Specific Inhibitors Biologically Active Natural Products Oxidosqualene ステロイドオキシドスクワレンラノステロール cDNA PCR ラットヒトクローニング

项目摘要

(1) The sequence covering the whole area of human lanosterol synthase was obtained in the previous year. Based on this sequence, the oligo NDAs corresponding to amino and carboxyl termini were synthesized and PCR was carried out to get full length clones. However, the expected product was not obtained. To get a full length clone, two overlapping fragments which had common restriction enzyme recognition site (SacI) in overlapping region, were amplified by PCR and combined after SacI digestion. The obtained full length fragment was ligated to multi-cloning site of yeast expression vector pYES2 in proper direction. The Erg7 deficient mutant of yeast was transformed by the resultant expression plasmid, and the functional complementation by the obtained clone was observed. And lanosterol synthase activity was detected in vitro using tritium labeled oxidosqualene as a substrate in cell free extract of transformed yeast. These results vigorously proved that the obtained clone is the gene encoding human lanosterol synthase. (2) In the similar manner as described above, pea cycloartenol synthase cDNA was also transferred into yeast expression vector and successfully expressed in the wild type yeast. (3) As for the screening of inhibitors from natural products, mutant yeast transformants with cDNAs of human and fungal (not yet cloned) lanosterol synthase were prepared. Simple comparison of growth rates of these transformants with test samples would indicate specific inhibitory activities of natural products contained in the samples. These specific inhibitors would serve as the leads for developping clinical druggs for hypercholestelemia and deep mycosis respectively.

(1)覆盖人羊毛甾醇合酶整个区域的序列是在前一年获得的。根据此序列，合成相应于氨基和羧基末端的寡核苷酸NDAs，并进行PCR以获得全长克隆。然而，没有得到预期的产品。为了获得全长克隆，通过PCR扩增在重叠区域中具有共同限制性内切酶识别位点（SacI）的两个重叠片段，经SacI酶切后合并。将获得的全长片段定向连接到酵母表达载体pYES 2的多克隆位点。用得到的表达质粒转化酵母的Erg 7缺陷突变体，观察所获得的克隆的功能互补。以氚标记的氧化角鲨烯为底物，对转化酵母的无细胞抽提物进行体外羊毛甾醇合成酶活性检测。这些结果有力地证明所获得的克隆是编码人羊毛甾醇合酶的基因。(2)以与上述类似的方式，豌豆环阿屯醇合酶cDNA也被转移到酵母表达载体中，并在野生型酵母中成功表达。(3)至于从天然产物中筛选抑制剂，制备了具有人和真菌（尚未克隆）羊毛甾醇合酶cDNA的突变酵母转化体。将这些转化体的生长速率与测试样品进行简单比较将表明样品中所含天然产物的特异性抑制活性。这些特异性抑制剂将分别作为开发高脂血症和深部真菌病临床药物的先导。