基于miRNA-155结构的人工miRNA表达载体的构建与评价

Construction and Evaluation of an Artificial miRNA Expression Vector Based on miRNA-155

目的:建立一种适用于人工miRNA(amiRNA)表达研究的克隆载体。方法:基于实验室构建的短发夹RNA(shRNA)表达载体pshOK-basic,将鼠源miRNA-155的侧翼序列插入合适的酶切位点构建得到amiRNA重组表达载体pOK-basic;应用本载体分别构建靶向萤火虫荧光素酶(luc2)和红色荧光蛋白(mCherry)基因的amiRNA并检测其沉默效果。结果:应用此载体能快速高效地构建amiRNA,靶向luc2和mCherry报告基因的amiRNA能较好地抑制靶基因的表达。结论:构建了一种能高效表达amiRNA的克隆载体,为amiRNA的进一步研究及应用奠定了基础。

Objective： To construct a novel vector which is suitable for artificial miRNA（amiRNA） expression study. Methods： Based on the laboratory-constructed shRNA expression vector pshOK-basic, the flanking sequenc- es of Mus musculus miRNA-155 were inserted into it in the appropriate restriction sites to build the amiRNA ex- pression vector pOK-basic. Three amiRNA targeting firefly luciferase（luc2） and red fluorescent protein（mCherry） were designed and cloned into this vector respectively, and their knockdown efficiency was detected. Results： The novel vector facilitated cloning amiRNA quickly and efficiently, and the amiRNA targeting luc2 and mCherry could inhibit target gene expression evidently. Conclusion： An efficient way for amiRNA clone was successfully established, and the novel vector pOK-basic provides us a useful tool for the further amiRNA research and application.