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)上一年获得了覆盖人羊毛甾醇合酶全区的序列。基于该序列，合成对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，没有得到预期的产物。为了获得全长克隆，通过PCR扩增两个在重叠区域具有共同限制性酶识别位点(SacI)的重叠片段，并在SacI消化后合并。将获得的全长片段以正确的方向连接至酵母表达载体pYES2的多克隆位点。用所得表达质粒转化酵母的Erg7缺陷突变体，并观察所得克隆的功能互补。并使用氚标记的氧化角鲨烯作为转化酵母无细胞提取物中的底物，在体外检测羊毛甾醇合酶活性。这些结果有力地证明了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，将豌豆环木菠萝烯醇合酶cDNA也转入酵母表达载体并在野生型酵母中成功表达。 (3)对于天然产物抑制剂的筛选，制备了带有人和真菌(尚未克隆)羊毛甾醇合酶cDNA的突变酵母转化体。这些转化体的生长速率与测试样品的简单比较将表明样品中所含天然产物的特定抑制活性。这些特异性抑制剂将成为开发分别治疗高胆固醇血症和深部真菌病的临床药物的先导药物。