反义RNA体外抑制丙型肝炎病毒基因表达的研究

Inhibition of hepatitis C virus gene expression by antisense nucleotide in vitro

目的 研究丙型肝炎病毒 (HCV)基因调控方式及反义RNA对HCV基因表达的体外抑制作用。方法 采用业已建立的HCV基因调控细胞模型 ,以重组质粒共转染策略 ,将反义RNA重组质粒与HCV 5′非翻译区 ( 5′UTR )调控的报道基因———虫荧光素酶 (luc)重组质粒共同转染人肝癌细胞系 (HepG2 ) ,并以不表达反义RNA的原核重组质粒和不含有HCV 5′UTR的luc重组质粒做为对照。转染细胞经短期培养后制备细胞提取液 ,荧光检测法检测luc基因的表达。结果 针对HCV 5′UTR的反义RNA可以在体外有效抑制由HCV 5′UTR调控的luc基因的表达 ,并具有剂量依赖效应。对照重组质粒转染实验证明 ,上述抑制作用呈序列特异性。结论 HCV 5′UTR具有调控下游目的基因表达的重要功能 ,反义RNA可以在体外有效抑制由HCV

Objective To study the mechanism of hepatitis C virus (HCV) gene regulation and the inhibitory effect of antisense RNA on HCV gene expression in vitro . Methods The hepatoblastoma cell line (HepG 2) was co-transfected by recombinant plasmid of antisense RNA complementary to HCV 5′ untranslated region (5′ UTR)and HCV 5′ UTR Directed luciferase (luc) gene expression recombinant plasmid. Meanwhile a reversed HCV 5′UTR recombinat plasnid which can not transcribe as antisense RNA in the cell and a recombinant plasmid in which the luc was regulated by simian virus 40 (sv40) 5′UTR were used as controls respectively. The level of luc gene expression was determined by an enzymatic assay. Results The antisense RNA which directed to HCV 5′UTR could obviously knock down the level of luc gene expression and the close-dependent inhibition of antisense RNA was observed at the same time. However the above inhibition was not shown in the cells co-transfected by reversed HCV 5′UTR recombinant plasmid and HCV 5′UTR directed luc gene expression recombinant plasmid. No reduction was obserred in luc gene expression level in the cell co-transfected by both antisense RNA recombinant plasmid and SV 40 5′UTR directed luc gene expression recombinant plasmid. Conclusion HCV 5′UTR plays an important role in regulation of viral gene expression. The antisense RNA complementary to HCV 5′UTR could effectively inhibit the gene expression regulated by HCV 5′UTR in vitro .