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目的 研究核酶在细胞外切割HCV的作用。方法 设计核酶cDNA序列。构建核酶重组质粒及HCV核心区基因cDNA重组质粒。分别进行体外转录 ,并加入γ 3 2 PATP以标记HCVRNA。将核酶RNA、HCV核心区RNA、RNA酶抑制剂及反应缓冲液混合温育 ,以核酶RNA切割HCVRNA。通过变性聚丙烯酰胺凝胶电泳及放射自显影来分析结果。结果 β 半乳糖苷酶表型筛选均可见蓝色及白色菌落生长 ;核酶重组质粒直接测序结果见预期核苷酸序列 ;HCV重组质粒限制性酶切分析见 410bp条带。核酶切割反应显示 :反应时间为 15和 30min时可见 45 3、30 7、146nt3条带 ;反应时间为 6 0min时仅见 30 7及 146nt2条带。结论 核酶重组质粒构建成功 ,所设计核酶对HCV有切割作用。
Objective To study the role of ribozyme in the cleavage of HCV extracellularly. Methods Design ribozyme cDNA sequence. Recombinant plasmid of ribozyme and HCV core region gene recombinant plasmid were constructed. Transcribed in vitro, respectively, and γ 3 2 PATP was added to label for HCV RNA. The ribozyme RNA, HCV core RNA, RNase inhibitor and reaction buffer were mixed and incubated, and RNAi was cleaved by ribozyme RNA. The results were analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. Results The β-galactosidase phenotypes showed blue and white colonies growth. The direct sequencing results of the ribozyme recombinant plasmids showed the expected nucleotide sequence. The 410 bp band was identified by restriction enzyme analysis of HCV recombinant plasmids. The ribozyme cleavage reaction showed 45 3,30 7,146nt3 bands when the reaction time was 15 and 30min and only 30 7 and 146nt2 bands when the reaction time was 60min. Conclusion The recombinant plasmid of ribozyme was successfully constructed, and the designed ribozyme cleaved the HCV.