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RNA干扰Neuropilins-2基因真核表达载体的构建及其鉴定

, PP. 691-694

Keywords: NRP,RNA干扰,真核表达载体

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Abstract:

目的构建Neuropilins-2(NRP2)基因RNA干扰(RNAi)的真核细胞表达载体。方法以NRP2为靶基因,以pGenSil-l质粒为载体,设计构件重组体,根据GenBank数据库提供的NRP2基因核苷酸序列,按照Tuschl设计原则,选择设计两条带发夹结构的核苷酸序列,克隆到空载体pGenSil-l中,转化DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析。重组pGenSil-NRP2载体转染LOVO细胞48h,收获细胞,采用Westernblotting检测其NRP2蛋白表达。结果经酶切鉴定筛选出的重组体测序结果与目的序列完全一致,重组载体构建成功。重组体pGenSil-NRP2转染LOVO细胞48h后NRP2蛋白表达显著降低。结论利用RNAi技术可成功构建抑制NRP2表达的小干扰RNA重组体。

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