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目的 :设计切割ki-rasG12V mRNA的特异性ribozyme(Rz2 17) ,明确其对癌基因ki-rasG12VmRNA的细胞内外切割活性 ,为以ki-rasG12VmRNA为特异性靶分子的基因治疗及癌基因ki-ras的功能研究提拱一种新的途径。方法 :依Symons总结的“锤头结构”原理 ,设计一种能特异性切割ki-rasG12VmRNA的ribozyme ,利用DNA重组技术构建ki-rasG12V外显子 1和ri-bozymeRz2l7的体外转录质粒及ribozymeRz2 17的真核表达质粒 ,体外转录获得ribozymeRz2 17及ki-rasG12V外显子 1mRNA ,在含Mg2 + 溶液中ribozymeRz2 17对其靶RNA分子进行切割。以RT -PCR对转染ribozymeRz2 17真核表达质粒的细胞ki-rasG12VmRNA进行半定量分析。结果 :ki-rasG12V外显子 1体外转录mRNA分子 ,能被ribozymeRz2 17定点切割而野生型ki-ras外显子 1体外转录mRNA则不被切割 ;转染ribozymeRz2 17的胰癌细胞ki-rasG12VmRNA含量减少 ,而转染ri bozymeRz2 17的肝癌细胞其内源性ki-rasmRNA含量无明显变化。结论 :ribozymeRz2 17无论在细胞内外均能剪切突变型ki-rasmRNA(G12V)而且其切割作用为突变型ki-rasG12VmRNA特异性的。
OBJECTIVE: To design a specific ribozyme (Rz217) for cleaving ki-rasG12V mRNA and to determine the intracellular and extracellular cleavage activity of the oncogene ki-rasG12V mRNA. The gene therapy and ki-ras gene therapy targeting ki-rasG12V mRNA as a specific target molecule Functional research Tigong a new way. Methods: According to the “Hammerhead Structure” principle summarized by Symons, a ribozyme capable of specifically cleaving ki-rasG12VmRNA was designed. In vitro transcribed plasmids of ki-rasG12V exon 1 and ri-bozymeRz2l7 and ribozymeRz2 17 Eukaryotic expression plasmid, in vitro transcription of ribozymeRz2 17 and ki-rasG12V exon 1 mRNA, ribozymeRz2 17 containing Mg2 + solution of its target RNA molecules were cut. Semi-quantitative analysis of the cell ki-rasG12V mRNA transfected with ribozymeRz2 17 eukaryotic expression plasmid was performed by RT-PCR. Results: In vitro transcription of ki-rasG12V exon 1 mRNA was detected by ribozymeRz217 site-directed mutagenesis and in vitro transcription of wild-type ki-ras exon 1 was not cleaved. Expression of ki-rasG12V mRNA in transfected ribozymeRz217 cells Decreased, while the ribozymeRz2 17 transfected liver cancer cells endogenous ki-rasmRNA content did not change significantly. Conclusion: ribozymeRz2 17 cleaves mutant ki-rasmRNA (G12V) both in vitro and in vivo and its cleavage is specific for mutant ki-rasG12V mRNA.