摘要
为了解丙型肝炎病毒(HCV)基因调控方式、探索反义寡脱氧核苷酸对丙型肝炎病毒基因表达的体外抑制作用。构建了由 HCV 5′非翻译区(5′UTR)翻译启动报道基因——虫荧光素酶(luc)基因的真核表达载体;人工合成了针对 HCV 5′UTR 及非结构蛋白5(NS5)区的反义寡脱氧核苷酸(ODN)。借助脂质体将 ODN 分别与所构建的载体—同转染人肝癌细胞系,短期培养后制备细胞提取液,以荧光发光法检测 luc 基因的表达。结果发现:两个针对 HCV 5′UTR 的 ODN 均可抑制 luc 基因的表达,其中与启动密码子上游序列(-21～-2)互补的反义 ODN 的作用更为明显,且呈剂量依赖性;与 NS5区互补的 ODN 不具有这种作用。本研究提示,HCV5′UTR 尤其是启动密码子上游序列对于其履行翻译启动功能至关重要。人工合成的反义 ODN 能够在细胞水平有效抑制 HCV 基因的表达。
In order to elucidate the role of hepatitis C virus 5′untranslated region(HCV 5′UTR)in HCV gene regulation and explore the inhibition of HCV gene expression by antisense oligodeoxynu- cleotides (ODN),antisense ODNs were synthesized against 5′untranslated region(5′ UTR) or nonstruc- tural region 5 (NS5)of HCV genome.A constructed vector which contained simian virus 40 promoter, HCV 5′UTR and luciferase (lue)gene were cotransfected into a hepatoblastoma cell line with each ODN. The production of active lue was monitored by an enzymtic assay.The results showed that two antisense ODNs complementary to the sequence of HCV 5′UTR can inhibit the expression of luc.The more effi- cant inhibition was observed in an antisense ODN which directed to the sequence immediately upstream authenic initiation codon.The dose dependent effect of this antisense ODN was also found with a maxi- mal inhibition of 99.0% at a concentration of 250μmol/L.The inhibition of antisense ODN complemen- tary to NS5 region had not been found.This study suggests that sequence which closed to authenic initia- tion codon in HCV 5′UTR may play an important role in the translation initiation of HCV RNA.The antisenes ODN might become apromising antiviral agent against HCV replication.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
1997年第4期190-193,共4页
Chinese Journal of Infectious Diseases
基金
国家自然科学基金资助
关键词
丙型肝炎病毒
基因表达
寡肿氧核苷酸
反义
Hepatitis C virus
Oligodeoxynucleotides
antisense
Gene expression regulation
