摘要
目的:分析丙型肝炎病毒(HCV)5′端非编码区(NCR)的结构域Ⅰ序列在其翻译启动活性中的作用。方法:以质粒pCMVN CRluc为模板,PCR扩增分别得到缺失5′端20nt和43nt的HCV 5′NCR片段,并分别替换pCMVNCRluc中的完整HCV 5′NCR,构建结构域Ⅰ缺失的HCV 5′NCR调控萤火虫荧光素酶(luc)基因表达的真核表达质粒(pCN1-d1、pCNl-d2)。以脂质体方法转染人肝癌细胞株HepG2,用双荧光素酶报告基因检测系统检测荧光素酶相对于内参考的海肾荧光素酶表达活性,同时采用RT-PCR方法检测转染后细胞中萤火虫荧光素酶基因的相对表达水平。结果:酶切和测序结果表明,各重组质粒构建成功。各重组质粒转染细胞后luc mRNA的相对表达水平与pCMVNRluc相比差异无显著性(P＞0．05);pCNl-dl、pCNl-d2表达的荧光素酶活性与pCMVNCRluc差异无显著性(P＞0．05)。结论:HCV 5′NCR的5′端20nt和43nt序列缺失不影响它的翻译启动活性。
Objective： Hepatitis C virus （HCV） infection is a major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma, which has been a worldwide health crisis. The fact that the highly conserved HCV 5＇non-coding region （5＇NCR） contains an internal ribosome entry site （IRES） essential for cap-independent translation of viral genes suggests that the HCV 5＇NCR is an attractive antiviral target. The objective of this experiment is to investigate the activity of structural domain I of HCV （5＇NCR） in translation initiation. Methods：The fragments of HCV 5＇NCR with deletions of 5＇-proximal 20 nucleotides and 43 nucleotides were amplified with polymerase chain reaction （PCR）, then replaced respectively the full length HCV 5＇NCR of plasmid pCMVNCRluc to construct two recombinant plasmids： pCNl-d1 and pCNl-d2, luciferase （luc） eukaryotic expression plasmids regulated by domain I deleted HCV 5＇NCR, and the recombinant plasmids were transfected into HepG2 cells with liposome transfection protocol, respectively. The relative luciferase activity was detected to analyze the regulatory effect of the truncted 5＇NCR on the luc gene expression, meanwhile the luc messenger RNA levels were detected by reverse polymerase chain reaction （RT-PCR）. Results：Restriction enzyme digestion and sequence analysis showed that the recombinant plasmids pCNl-d1 and pCNl-d2 were successfully constructed. Semi-quandfied RT-PCR demonstrated the luc mRNA levels were not significandy different （P〉0.05）, pCNI-d1 and pCN1-d2 expression of luc in transefected cells are 0.8702±0.0206 and 0.8842 ±0.0413 respectively. There were also no significant difference among the relative luciferase activity of plasmids pCNl-d1, pCNl-d2 and pCMVNCRluc （P〉0.05）： 0.80±0.08, 0.71±0.04 and 0.70±0.10, respectively. Conclusion：Deletions of 5＇-proximal 20 and 43 nucleotides of HCV 5＇NCR show no effect on its translation initiation activity. It indicates that the structural domain I has no effec
出处
《现代生物医学进展》
CAS
2007年第5期699-701,共3页
Progress in Modern Biomedicine
基金
湖南省医药卫生科技项目(B2004-011)
关键词
丙型肝炎病毒
荧光素酶
翻译启动活性
Heptitis C virus （HCV）
Luciferase
Translation initiation activity
