丙肝病毒核心基因克隆载体pGEM-HCV/core的构建

Construction of cloning plasmid pGEM-HCV/core containing the core gene of HCV

目的构建丙肝病毒核心基因克隆载体pGEM-HCV/core,为进一步研究外部引导序列(external guide sequence,EGS)引导核糖核酸酶P在RNA水平阻断丙肝病毒核心基因的表达做好前期基础。方法将含HCV全长基因的pBRTM/HCV1-301l质粒于大肠杆菌JMl09内扩增;提取pBRTM/HCV1-301l质粒;从pBRTM/HCV1-301l质粒中PCR扩增出HCV核心基因片段并将其插入pGEM-3Z克隆载体;以得到重组的pGEM-HCV/core克隆载体;最后EcoR I and Hind III双酶切鉴定HCV核心基因克隆载体。结果pGEM-HCV/core克隆载体经双酶切、凝胶电泳证明插入片段与HCV核心基因片段大小相符;经测序证明其插入序列与HCV core基因序列一致。结论成功构建了HCV核心基因的克隆载体pGEM-HCV/core。

Objective To construct the clone plasmid containing the core gene of HCV, preparing for the research of blockage of the expression of HCV core gene guided by EGS at the level of mRNA. Methods After expanding plasmid pBRTM/HCV1-3011 （ containing the whole genome of HCV） through E. coli JM109. The core gene with pBRTM/HCV1-3011 is amplified with PCR as the template; The core segment was inserted into the clone plasmid pGEM-3Z. Plasmid pGEM - HCV/core was digested with EcoR I and Hind III. Results The PCR result of HCV core has correct size and sequences. Conclusion The clone plasmid pGEM-HCV/core has successfully been constructed.