锤头状核酶对长底物mdr1 mRNA的体外切割

Ribozyme-mediated cleavage of long mdr1 mRNA

应用计算机对ｍｄｒ１ｍＲＮＡ二级结构进行模拟，设计针对ｍｄｒ１ｍＲＮＡ１９５９位ＧＵＣ的“锤头状（Ｈａｍｍｅｒｈｅａｄ）”核酶（Ｒｉｂｏｚｙｍｅ１，ＲＺ１）基因定点克隆于质粒ｐＧＥＭＥＸ１的ＢａｍＨⅠ和ＥｃｏＲⅠ位点上；将ｍｄｒ１ｃＤＮＡ１３８３ｂｐ的片段亚克隆于ｐＧＥＭＥＸ１的ＥｃｏＲⅠ位点上；在Ｔ３起动子作用下体外分别转录成ＲＺ１和１３８３ｎｔ的ｍｄｒ１ｍＲＮＡ，ＲＺ１在体外将１３８３ｎｔ的ｍｄｒ１ｍＲＮＡ切割成两个片段。

A hammerhead ribozyme has been designed to cleave the GUC sequence at codon 1959 of mdr1 mRNA after analysis of the secondary structure of mdr1 mRNA by computer.The hammerhead ribozyme 1 gene was synthesized and was cloned into the BamH I/EcoR I sites of pGEMEX1,named pEXRZ1.The 1383bp fragment of mdr1 mRNA was subcloned into the EcoR I site of pGEMEX1,named pMDR1383 (+5).The pEXRZ1 and pMDR 1383 (+5) were lineralized with BamH I and transcribed with T3 promotor in vitro.The ribozyme 1 showed strong cleavage activity after mixed the 2 transcripts for 2 hours at 42℃ and 52℃.The ribozyme designed in this study may be used as a tool to reverse multidrug resistance.