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二价核酶基因构建及对PLRV负链靶序列的体外切割研究

DOI: 10.3969/j.issn.1000-7091.2012.02.003, PP. 12-18

Keywords: 二价核酶,马铃薯卷叶病毒,复制酶基因,体外切割

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Abstract:

根据锤头状核酶的作用模式,设计、合成并克隆了特异性切割马铃薯卷叶病毒(Potatoleafrollvirus,PLRV)中国分离株(PLRV-Ch)复制酶基因负链RNA的二价核酶序列。将该核酶基因克隆到pGEM-4Z中,构建成体外转录载体pGEM-4ZDR;同时将包含核酶切割识别位点的PLRV-Ch复制酶基因cDNA近5’端的874bp片段反向插入到pGEM-4Z中,构建了体外转录载体pGEM-4ZR5。以线性化的重组质粒pGEM-4ZDR和pGEM-4ZR5为模板,在T7RNA聚合酶的作用下,分别转录获得核酶RNA和PLRV-Ch复制酶基因5’端负链RNA底物片段。将以上2种RNA转录物混合并经37℃保温后,检测结果表明,所得核酶RNA对PLRV-Ch复制酶基因负链RNA在体外具有较强的特异切割活性。

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