针对不同基因靶位的锤头状核酶对HBV的抑制作用研究

Inhibition of Hepatitis B Virus Expression and Replication in Cultured Cells by Hammerhead Ribozymes Targeted at Different Sites

前基因组mRNA是HBV(HepatitisBvirus)基因表达和复制的重要中间产物,全长的前基因组mRNA分子具有复杂易变的二级结构,是设计抑制HBV的核酶时所必须考虑的因素。我们使用多个最新的计算机软件对HBV前基因组mRNA二级结构进行模拟、分析,在全面分析核酶的可作用位点的基础上设计三个针对不同基因靶位的锤头状核酶,并对它们在细胞中对HBV的抑制作用进行研究。结果表明在HBV前基因组mRNA上存在几个高度复杂二级结构的区域,可能对核酶完全不敏感,而S、C、X基因的编码区是合适的核酶作用位点,都可达到对HBV的有效抑制,而且X基因位点的核酶对HBV的各种mRNA的抑制作用最为明显,是设计针对HBV核酶时应该优先考虑的位点。

The pregenomic RNA, serves as a template for reverse transcription and encodes the reverse transcriptase for Hepatitis B virus (HBV) replication, has the energetically flexible secondary and tertiary structures, which may reduce the accessibility of ribozyme. To identify accessible target sites for hammerhead ribozyme, the secondary structure analysis of the full-length HBV pregenomic RNA was analyzed using the newest bioinformatic tools. Screening the accessible sites for ribozyme based on the structures show that the accessible sites located on the encode region of S, C, X genes and almost not on the regulation region or the encapsidation signal(ε). Thereby three hammerhead ribozymes targeted different sites were designed and transfected into cells to evaluate the catalytically inactivation of HBV gene expression. Experimental results validated that the encoding regions of S, C, X genes were the effective cleavage sites for hammerhead ribozyme and the ribozyme targeted X gene was most potential to inhibit HBV RNA accu- mulation.