位点选择对核酶体外切割活性的影响

INFLUENCE OF CLEAVAGE SITE SELECTION ON RIBOZYMEMEDIATED CLEAVAGE OF MDR1 mRNA

应用计算机对ｍｄｒ１ｍＲＮＡ二级结构进行模拟，设计针对ｍｄｒ１ｍＲＮＡ１９５９位ＧＵＣ和ｍｄｒ１ｍＲ－ＮＡ１７０５位ＧＵＵ的锤头状（Ｈａｍｍｅｒｈｅａｄ）核酶（Ｒｉｂｏｚｙｍｅ）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起动子作用下体外转录成ＲＺ１、ＲＺ２和１４３０ｎｔ的ｍｄｒ１ｍＲＮＡ，比较ＲＺ１和ＲＺ２对１４３０ｎｔ的ｍｄｒ１ｍＲＮＡ的切割活性的差异，结果ＲＺ１对１４３０ｎｔ的ｍｄｒ１ｍＲＮＡ切割活性较ＲＺ２高得多。上述结果表明：在应用核酶技术进行基因治疗时，应选择有效的核酶切割位点，以达到有效的治疗目的。

Two hammerhead ribozymes have been designed to cleave the GUC sequence at codon 1959 and GUU at codon 1705 of mdrl mRNA according to the analysis of the secondary structure of mdrl mRNA with computer.The hammerhead ribozyme genes were synthesed and cloned into the BamH I/EcoR I sites of pGEMEX1,named pEXRZ1 and pEXRZ2.The 1383bp fragment of mdrl cDNA was subcloned into the EcoR I site of pGEMEX1,named pMDR1383(+5).The pEXRZ1 and pEXRZ2 as well as pMDR 1383(+5)were lineralized with BamH I and transcribed with T3 promotor in vitro.The ribozyme 1 showed strong cleavage activity than the ribozyme 2 after mixed the transcripts for 2 hours at temperature 42 °C and 52 °C.The results imply that cleavage site selection is an important factor that influences the cleavage activities of ribozymes.