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目的构建含丙型肝炎病毒(HCV)核糖体插入位点(IRES)序列的基因克隆,为以后的亚克隆和抑制肝炎病毒作用的研究提供实验材料。方法以含HCV全长基因的质粒为模板,用PCR技术扩增出HCV的IRES序列,将扩增产物IRES基因插入到PMD18T载体后转化DH5α,筛选阳性克隆,抽提重组质粒并进行PCR及酶切鉴定,再行序列分析。结果经PCR获得355bp含限制性内切酶位点的阳性产物,T载体克隆、PCR及酶切鉴定和序列分析后证实,克隆片段与GeneBank中该基因的序列同源性为99%。结论该实验成功构建了含HCV IRES基因序列的T载体克隆,提示该克隆是用作亚克隆和抑制肝炎病毒研究的理想克隆。
Objective To construct a gene clone containing the ribosomal insertion site (IRES) of hepatitis C virus (HCV), and to provide experimental materials for the subsequent subcloning and inhibition of hepatitis virus. Methods The HCV full-length cDNA sequence was amplified by PCR using the plasmid containing HCV full-length gene as a template. The amplified product IRES gene was inserted into PMD18T vector and transformed into DH5α. The positive clones were screened and the recombinant plasmids were extracted and subjected to PCR and enzyme Cut identification, and then sequence analysis. Results The positive product of 355bp restriction site was obtained by PCR. The T vector was cloned, confirmed by PCR, restriction enzyme digestion and sequence analysis. The sequence homology between the cloned fragment and GeneBank was 99%. Conclusion This experiment successfully constructed T vector clones containing HCV IRES gene sequence, suggesting that this clone is an ideal cloning for subcloning and inhibiting hepatitis virus.