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    • 7. 发明授权
    • Sterol .DELTA.14 reductase screen
    • Sterol DELTA 14还原酶屏
    • US5591576A
    • 1997-01-07
    • US107348
    • 1993-08-16
    • Margaret H. K. LaiDonald R. KirschMartin Bard
    • Margaret H. K. LaiDonald R. KirschMartin Bard
    • C12N9/02C12N15/53C12Q1/18C12Q1/26C12N15/63C12Q1/02C12Q1/68
    • C12N9/001C12Q1/18C12Q1/26C12Y103/0107
    • A binary assay identifies agents that inhibit sterol .DELTA.14 reductase involved in ergosterol biosynthesis. In the primary screen, sterol .DELTA.14 reductase inhibition by a test sample is assayed by adding the test sample to a culture of Neurospora crassa having an erg-3 mutation and also to a culture of a strain having an erg-1 mutation, comparing the extent of growth inhibition after incubation in the two cultures, and identifying as positives those samples that show growth inhibition in the erg-3 culture exceeding that in the erg-1 culture. In the secondary screen, samples that test positive in the primary screen are reassayed by adding the test sample to a culture of a Saccharomyces cerevisiae strain into which has been introduced multiple copies of a gene encoding sterol .DELTA.14 reductase and also to a strain of S. cerevisiae that does not have the introduced gene; positive samples are identified after incubation by observation that growth inhibition in the culture having no introduced reductase gene exceeds growth inhibition in the culture having the introduced reductase gene. In preferred embodiments, a known inhibitor of sterol .DELTA.14 reductase is employed in solidified media in both the primary and the secondary screens, resulting in an assay that is highly sensitive and specific for the detection of sterol .DELTA.14 reductase inhibitors.
    • 二元测定法鉴定了抑制参与麦角固醇生物合成的甾醇DELTA14还原酶的药剂。 在主筛网中,通过将测试样品加入到具有erg-3突变的粗糙脉孢菌培养物中,并将其与具有erg-1突变的菌株的培养物进行比较,来测定测试样品的固醇DELTA 14还原酶抑制 在两种培养物中孵育后的生长抑制程度,并且鉴定出在erg-3培养物中显示生长抑制的样品超过erg-1培养物中的那些样品的阳性。 在次级屏幕中,通过将测试样品加入到已经引入了多拷贝的编码固醇DELTA14还原酶的基因的Saccharomyces cerevisiae菌株的培养物中,并且还添加到S的菌株 没有引入基因的酿酒酵母; 通过观察,通过观察到没有引入的还原酶基因的培养物中的生长抑制超过在具有引入的还原酶基因的培养物中的生长抑制的情况下,鉴定阳性样品。 在优选的实施方案中,在初级和次级筛中的固化培养基中使用已知的甾醇DELTA 14还原酶抑制剂,导致对检测固醇DELTA14还原酶抑制剂具有高度敏感性和特异性的测定。