Inouye S, Tsuji F I
Yokohama Research Center, Chisso Corporation, Kanagawa, Japan.
Anal Biochem. 1992 Feb 14;201(1):114-8. doi: 10.1016/0003-2697(92)90182-7.
A luminescence method for monitoring gene expression in Chinese hamster ovary cells using apoaequorin as a secreted reporter enzyme is described. In this method, the cell is not disrupted prior to assay as in the earlier aequorin procedure and in the firefly method. The apoaequorin secretion vector is constructed by fusing the DNA fragment of the signal peptide sequence of human follistatin to the apoaequorin gene. Transfection of Chinese hamster ovary cells with the vector causes the apoaequorin to be secreted directly into the culture medium. Assay is carried out by removing a small aliquot of the culture medium, incubating it with coelenterazine, and adding Ca2+ to trigger light emission from the regenerated aequorin. The light intensity is measured with a photomultiplier photometer and is proportional to the amount of apoaequorin present. The method is highly specific and sensitive and can be carried out in a relatively short period of time.
描述了一种使用脱辅基水母发光蛋白作为分泌型报告酶来监测中国仓鼠卵巢细胞中基因表达的发光方法。在该方法中，与早期的水母发光蛋白程序和萤火虫方法不同，在测定之前细胞不会被破坏。通过将人卵泡抑素信号肽序列的DNA片段与脱辅基水母发光蛋白基因融合来构建脱辅基水母发光蛋白分泌载体。用该载体转染中国仓鼠卵巢细胞会使脱辅基水母发光蛋白直接分泌到培养基中。测定时，取一小份培养基，与腔肠素一起孵育，然后加入Ca2+以触发再生的水母发光蛋白发光。用光倍增管光度计测量光强度，其与存在的脱辅基水母发光蛋白量成正比。该方法具有高度特异性和敏感性，并且可以在相对较短的时间内完成。
