建立转录丙型肝炎病毒结构区基因细胞模型的研究

Study on establishment of cellular model transcripting HCV structural genes

目的 :建立转录丙型肝炎病毒 (HCV)结构区基因的细胞模型 ,为研究中药在体外对HCV结构区基因转录的抑制作用提供实验用细胞模型。方法 :用脂质体将已构建好的含HCV结构区基因 1.73kb的真核细胞表达载体pBK CMV转入人HeLa细胞 ,用G418选择性培养基筛选出成功转染的HeLa细胞 (HeLaD细胞 ) ,用Trizol提取HeLaD细胞的总RNA ,通过特异性引物进行逆转录 聚合酶链反应 (RT PCR) ,从mRNA水平来验证HeLaD细胞中HCV结构区基因的有效转录。结果 :对从HeLaD细胞抽提的总RNA作RT PCR ,得到 14 4bp的特异的扩增片段 ,证实HCV结构区基因在HeLaD细胞中有转录水平的表达。结论 :重组质粒pBK CMV能被成功地转染人HeLa细胞 ,且HCV的结构区基因能够有效地表达 ,成功建立了能转录HCV结构区基因的细胞模型 HeLaD。

Objective In order to study the effect of Chinese herbs' on the transcription of hepatitis C virus (HCV) structural genes,the cell model being able to transcript the HCV structural genes was established.Methods Using lipofectin,human HeLa cells were transfected with the eukaryotic expression vector pBK-CMV which contained HCV structural gene fragments 1.73-kb.The selective medium containing G418(600-mg·L -1) was used to select the correctly trasfected HeLa cells that was named as HeLaD.Then the total RNA of the transfected HeLa cells was extracted by Trizol reagent and RT-PCR assays were used to further verify the HCV structural gene fragments in mRNA level.Results The RT-PCR results showed that a specific product of 144-bp DNA fragment could be seen in HeLa-D cells transfected with the recombinant plasmid pBK-CMV indicating that the HeLa-D cells could transcript the HCV structural genes successfully.Conclusion Human HeLa cells can be transfected with the HCV structural genes.Further more,the HCV genes can be expressed and transcripted successfully in the HeLa-D cells.