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设计合成针对丙型肝炎病毒 (HCV )核心区基因 (第3 84— 3 86nt)的锤头状核酶 (Ribozyme ,Rz) ,从丙型肝炎病人的血清中提取HCV RNA ,经RT—PCR扩增HCV基因片段( 4 12bp) ,作为核酶的靶序列 ,将核酶基因和HCV靶基因片段分别克隆人反转录病毒载体 pLXN中构建重组载体 ,并用电击法分别转入包装细胞PA3 17中 ,将上述两种转细胞释放到培养液中的含Rz的假病毒颗粒和含HCV靶基因的假病毒颗粒混合后 ,共同感染空白的PA3 17细胞 ,被感染细胞维持液的RT -PCR结果表明 ,无HCV靶基因产物即无靶基因的假病毒颗粒释放至维持液中 ,证明Rz在细胞内有切割HCV靶RNA的活性。
Design and synthesis of Ribozyme (Rz) targeting HCV core region gene (3 84-386 nt), extract HCV RNA from the serum of hepatitis C patients and amplify by RT-PCR The HCV gene fragment (4 12bp) was amplified and used as target sequence of ribozyme. The ribozyme gene and HCV target gene fragment were respectively cloned into the human retroviral vector pLXN to construct the recombinant vector, and then transferred into the packaging cell PA3 17 by electroporation , The Rz-containing pseudovirions released into the culture medium and the pseudovirions containing the HCV target gene were mixed together to co-infection with blank PA3 17 cells, and RT-PCR results of infected cell maintenance liquid , And the non-HCV target gene product, ie, the dummy gene without target gene, was released into the maintenance solution, demonstrating that Rz has an activity of cleaving HCV target RNA in the cell.