PHOTOPROTEIN AEQUORIN - USE AS A REPORTER ENZYME IN STUDYING GENE-EXPRESSION IN MAMMALIAN-CELLS
PHOTOPROTEIN AEQUORIN - USE AS A REPORTER ENZYME IN STUDYING GENE-EXPRESSION IN MAMMALIAN-CELLS
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DOI：
10.1016/0378-1119(90)90260-x
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发表时间：
1990-12-15
期刊：
影响因子：
3.5
通讯作者：
SAKAKI, Y
中科院分区：
文献类型：
--
作者：
TANAHASHI, H;ITO, T;SAKAKI, Y
Aequorin是存在于水母Aequoria Victoria中的一种发光蛋白质，它在钙离子存在的情况下发出光(波长为460 nm)。我们在此报道了Aequorin可以作为一种报告酶来监测真核细胞中的基因表达。将Apoaequorin基因与SV40、RSV和HSV-1 tk基因启动子融合，并导入CV-1、COS、HeLa等细胞中。在转染后的适当时间，用光电倍增管光度计监测钙离子触发的460 nm处的光的强度，以测量细胞提取物中的水通道蛋白活性。Aequorin法与传统的CAT法一样灵敏，用Aequorin法估计的各种启动子的相对活性与用CAT法得到的结果基本一致。从提取液的制备到活性的测定，不需要使用放射性化合物，可以在6-7小时内完成水乙醇苷的测定。
Aequorin is a luminescent protein present in the jellyfish Aequoria victoria which emits light (at 460 nm) in the presence of Ca2+. We report here that aequorin can be used as a reporter enzyme to monitor gene expression in eukaryotic cells. A cDNA encoding apoaequorin was fused to several eukaryotic promoters, including those of SV40, RSV and the HSV-1 tk gene, and introduced into several cell lines such as CV-1, COS and HeLa. At appropriate times after transfection, the aequorin activities in cell extracts were measured by monitoring the intensity of light emitted at 460 nm when triggered by Ca2+ by the use of a photomultiplier photometer. The aequorin assay was shown to be as sensitive as the conventional CAT assay, and the relative activities of various promoters estimated by the aequorin assay were in general agreement with those obtained by the CAT assay. The aequorin assay can be done within 6-7 h from the preparation of extract to the measurement of activity without using radioactive compounds.