丙型肝炎病毒5’端非编码区的5’端序列对其翻译启动活性的影响  

Effect of the 5'-proximal sequence of HCV 5'NCR on its translation initiation activity

作　　者：刘水平[1] 赵俊琴[1] 谭德明[2] 朱海鹏[2] 余俊龙[1] 

机构地区：[1]中南大学湘雅医学院微生物学教研室,长沙410078 [2]中南大学湘雅医院传染病研究所

出　　处：《胃肠病学和肝病学杂志》2005年第4期355-358,共4页Chinese Journal of Gastroenterology and Hepatology

基　　金：湖南省科技计划项目(02JTY3003)

摘　　要：目的分析丙型肝炎病毒(HCV)5’端非编码区(5’NCR)的5’端序列对其翻译启动活性的影响。方法用PCR技术扩增获得全长和5’端17个碱基缺失的HCV5’NCR片段,定向克隆至表达质粒pSEAP2Control的SEAP基因上游,分别构建成全长和5’端截短的HCV5’NCR调控SEAP表达的重组质粒pNCRSEAP和pdNCRSEAP。用脂质体方法将重组质粒转染至肝细胞株QSG7701,并用化学发光法检测SEAP的表达。结果酶切和测序结果表明,重组质粒pNCRSEAP和pdNCRSEAP构建成功。表达的发光强度分别为390482±42856和635265±52285RLU,二者有差异显著性(P<0.01)。结论HCV5’NCR的5’端碱基缺失提高了它对SEAP基因的翻译启动活性,为进一步分析HCVIRES的结构提供了实验基础。Objective To compare the translation initiation activity between full-length HCV 5＇NCR and its 5＇-terminal truncated version .Methods The fragments of full-length HCV 5＇ noncoding region （5＇ NCR ）and truncated HCV 5＇ NCR with deletion of the 5 ＇-proximal 17 nucleotides were amplified with PCR, and were fused in-frame into immediate upstream of SEAP gene of pSEAP2-Control, a SEAP eukaryotic expression plasmid, to generate recombinant plasmids pNCRSEAP and pdNCRSEAP respectively, which were confirmed with enzymatic digestion and DNA sequencing. With the liposome transfection technique, the recombinant plasmids were transfected into hepatocytes QSG7701, and the SEAP activity of cell culture media was monitored quantitatively by the chemiluminescent method. Results The recombinant plasmids were successfully constructed. The light emission intensity of pNCRSEAP and pdNCRSEAP expression was 390482 ＋ 42856 and 635265 4 ＋ 52285 RLU respectively. There was significant difference between them（ P 〈 0.01 ）. Conclusion Deletion of the 5 ＇-proximal 17nucleotides of HCV 5＇NCR can enhance its translation initiation activity for SEAP gene, and lay experimental foundation for further study on the structure of HCV IRES.

关 键 词：肝炎病毒 丙型 5’端非编码区 内部核糖体进入位点 翻译启动活性 

分 类 号：R373.2[医药卫生—病原生物学]