锤头状核酶对不同长度mdrl mRNA的体外切割

Hammerhead ribozyme-mediated cleavage of mdrl mRNA with different length

目的：探讨膀胱癌多药耐药性逆转的新方法。方法：应用计算机对mdrlmRNA二级结构进行模拟，设计针对mdrlmRNA1959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mdrlcDNA864bp的片段亚克隆于pGEMEX-1的HindⅢ和EcoRⅠ位点上，mdrlcDNA1383bp片段亚克隆于pGEMEX-1的EcoRⅠ位点上，在T3启动子作用下体外转录成RZ1、864nt和1430nt的mdrlmRNA。结果：RZ1在体外分别将864nt和I430nt的mdrlmRNA切割成两个片段，切割温度在42℃和52℃两个条件下切割效率差异不明显。结论：核酶对底物的体外切割活性取决于核酶切割位点的选择，与底物长度无关。

Objective: To explore the new methods to reverse the multidrug resistance of bladder cancer.Methods: A hammerhead ribozyme was designed to cleave the GUC sequence at codon 1959 of mdrl mRNAafter the analysis of the secondary structure of mdrl mRNA by computer. The hammerhead ribozyme 1 genewas synthesized and cloned into the BamH I /EcoR I sites of pGEMEX-1 called pEX-RZl. The 864 bp fragment of mdrl cDNA was subcloned into the Hnd, /EcoR I sites of pGEMEX-1 called pMDR-864. The1 383 bp fragment of mdrl cDNA was subcloned into the EcoR I site of pGEMEX-1 called pMDR-1383 (+5).The pEX-RZl and pMDR-864 were lineralized with Hnd Ⅲ while the pMDR-1383(+5) was lineralized withBam H I and these lineralized plasmids were transcribed with T3 promotor in vitro. Results: The ribozyme 1showed strong cleavage activity after heing mixed with the transcribed RZl and its substrates for 2 hours at42℃ and 52℃ Concluslon: The designed ribozyme can be used as a tool to reverse the multidrug resistanceof bladder cancer. the cleavage activity of the ribozyme is not associated with the length of its substrate butdepended on the location of the cutting site.