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为了进行以基因为靶的抗丙型肝炎病毒(HCV)药物的筛选及评价,将HCV5＇NCR-C基因与荧光素酶基因的融合基因片段插入pCI-neo表达载体,通过PCR扩增、酶切反应及荧光素酶瞬间表达活性鉴定,获得HCV 5＇NCR调控荧光素酶的稳定表达质粒pHCV-neo4,将其转染HepG2细胞,经C418筛选,从150个克隆中获得一个高荧光素酶活性表达的细胞株HepG2.9706.该株细胞内HCV片段的DNA及mRNA检测均呈阳性,转基因拷贝数为140.利用该细胞株,通过脂质体介导,对3条针对HCV调控基因的反义寡核苷酸(ASODNs)即HCV363,HCV349,HCV279进行了活性评价,结果显示,三种ASODNs均具有特异性的剂量依赖性抑制活性,浓度在0.5μmol/L时,三者对HCV 5＇NCR调控荧光素酶表达抑制率分别为61％,55％及48％.该研究提示反义寡核苷酸有可能成为治疗HCV的新途径.
For screening and evaluation of gene-targeted anti-hepatitis C virus (HCV) drugs, the fusion gene fragment of HCV 5 ’NCR-C gene and luciferase gene was inserted into pCI-neo expression vector, Cut reaction and transient expression of luciferase activity to obtain HCV 5’NCR regulatory luciferase expression plasmid pHCV-neo4, the transfected HepG2 cells, screened by C418, obtained from 150 clones of a high luciferase The active cell line HepG2.9706 was used to detect the DNA and mRNA of the intracellular HCV fragment.The transgene copy number was 140. By using this cell line, The antisense oligonucleotides (ASODNs), HCV363, HCV349 and HCV279, were evaluated for activity. The results showed that all three ASODNs had specific dose-dependent inhibitory activities. When the concentration of ASODNs was 0.5μmol / L, The inhibitory rates of NCR-regulated luciferase expression were 61%, 55% and 48%, respectively, suggesting that antisense oligonucleotides may be a new therapeutic approach for HCV.