丙肝病毒核心基因靶向性M1GS 核酶的体外抗病毒活性

Determination of the in vitro antiviral activity of an engineered M1GS ribozyme that targets to the core gene of hepatitis C virus

摘要:

摘要:【目的】丙型肝炎病毒(Hepatitis C Virus，HCV)是引起病毒性肝炎的重要病原。目前临床HCV 感染多采用干扰素联合病毒唑进行治疗，但其应答率低且感染易反复，探索新型抗HCV 治疗策略与药物具有紧迫的现实意义。【方法】基于大肠埃希菌来源的核糖核酸酶P(RNase P)，针对HCV 核心基因的序列，设计一小段与之互补的外部引导序列(Guide Sequence，GS)，通过PCR 将其共价连接至大肠埃希菌RNase P 催化性亚基(M1 RNA)的3'末端，从而构建一种靶向性的核酶———M1GS。【结果】构建的M1GS-HCV/C52核酶在体外可对靶RNA片段产生特异性切割;在HCV 感染的Huh7.5.1细胞，该人工核酶也能够显著抑制HCV核心基因的表达，并使培养上清中HCV RNA的拷贝数减少约1500倍。【结论】本研究构建的M1GS-HCV/C52核酶具有显著的体外抗病毒活性，从而为HCV的治疗研究提供了一条潜在途径。

关键词:关键词:丙型肝炎病毒，核心基因，M1GS 核酶，抗病毒

Abstract:

Abstract:［Objective］Hepatitis C virus (HCV) is one of the major pathogens that lead to viral hepatitis． At present，Interferon treatment in combination with ribavirin is the first line clinical therapeutic approach． However，the responses are usually poor and the viral infection reoccurs． Therefore，exploring new antiviral agents and therapies is under urgent needs．［Methods］The sequence and structure of the core coding region of HCV genome were analyzed through the two computer software，DNAMAN and RNA Structure． The cytosine 52 nt downstream of the AUG initiation triplet was identified as the optimal target cleavage site． Based on the flanking sequence of this assumed cleavage site，a guide sequence (GS) was designed and covalently linked to the 3 prime terminus of the M1 RNA，which is catalytic subunit of the RNase P derived from Escherichia coli using PCR． We named this new targeting ribozyme M1GS-HCV /C52 and it antiviral activities were analyzed in cultured cells．［Results］In the in vitro cleavage assay，M1GS-HCV /C52 ribozyme could effectively cleave the HCV target RNA into two fragments at the specific cleavage site． Moreover，comparing to the blank control，this engineered M1GS ribozyme could reduce the core protein expression of more than 80% in the HCVinfected host cell and lead to a 1500-fold reduction of HCV RNA copies in the culture supernatant． An another M1GS ribozyme，M1GS-HCV /C52 * ，which has the same guide sequence but does not contain a 24nt-long bridge sequence，did not exhibit apparent inhibition for the expression of HCV core gene and viral proliferation in our paralleled assay．［Conclusion］We successfully constructed an M1GS ribozyme showing affective and specific cleavage of target viral RNA．Further results showed that the engineering ribozyme had notably antiviral activity in cultured cells，thus provided a new promising approach for clinical anti-HCV therapeutic strategy.

Key words:Keywords: Hepatitis C virus (HCV)，core gene，M1GS ribozyme，antiviral