M1GS-HCV/C141核酶的构建及其体外抗病毒活性测定

Construction of an engineered M1GS-HCV/C141 ribozyme and determination of its antiviral activity in vitro

摘要:

丙型肝炎病毒 (Hepatitis C virus，HCV) 是引起慢性肝炎的重要病因之一，严重危害公众健康。目前临床HCV感染常采用干扰素联合病毒唑进行治疗，然而应答率不高且易反复。因此，探索新型抗HCV治疗策略及药物显得尤为迫切。针对HCV核心基因的序列，设计与之互补的引导序列 (Guide Sequence，GS)，通过PCR的方法将其共价连接于大肠杆菌核糖核酸酶P(RNase P) 催化性亚基 (M1 RNA) 的3￠末端，成功构建了一种靶向性M1GS核酶 (M1GS-HCV/C141)。经体外切割试验、胞内反义效应及胞内毒性研究，结果表明：M1GS-HCV/C141核酶不仅能够在体外对靶RNA片段产生特异性切割，在HCV感染的Huh7.5.1细胞中，也能显著抑制病毒核心蛋白的表达，进而使HCV RNA的拷贝数减少约1 000倍。因此，文中构建的M1GS-HCV/C141核酶在体外具有显著的抗HCV活性，这为HCV治疗研究提供了一条新的潜在途径。

Abstract:

Hepatitis C virus (HCV), one of the major pathogens of viral hepatitis, causes significant hazards in humans. Interferon treatment in combination with ribavirin is used as the first line clinical treatment for HCV infection. However, good response to this treatment has only been observed in few patients and repeated recurrence has also been reported frequently. Therefore, new antiviral agents and therapies are in urgent demand. Here, we report a newly constructed Escherichia coli RNase P based M1GS ribozyme that can specifically and efficiently target the core gene of HCV. The guide sequence (GS) of this MIGS was designed according to the sequence of the core coding region of HCV genome. The GS was then covalently linked to the 3' terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. The specification of this sequence-specific ribozyme, M1GS, was then examined using an in vitro cleavage assay. The cytotoxicity and its activity in inhibition of HCV gene expression and viral proliferation were further studied in vivo. Our results show that the reconstructed M1GS ribozyme displayed obvious catalytic activity in cleaving target mRNAs fragment in vitro. Notable reduction in the expression of HCV core protein and a 1 000-fold reduction in viral growth were also observed in cultured HCV infected Huh7.5.1 cells expressing the functional M1GS ribozyme. This study demonstrated a direct evidence for the antiviral activity of the customized M1GS-HCV/C141 ribozyme, and thus provided a promising new strategy for clinical treatment of HCV infection.