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Inhibition of the replication of grass carp reovirus in CIK cells with plasmid-transcribed shRNAs

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文献类型：外文期刊

作者： Ma, Jie ² ; Wang, Weiming ² ; Zeng, Lingbing ¹ ; Fan, Yuding ¹ ; Xu, Jin ¹ ; Zhou, Yong ¹ ;

作者机构： 1.Chinese Acad Fishery Sci, Div Fish Pathol, Yangtze River Fisheries Res Inst, Wuhan 430223, Hubei, Peoples R China

2.Huazhong Agr Univ, Coll Fisheries, Wuhan 430071, Peoples R China

关键词： DNA;plasmid-transcribed shRNA;viral replication

期刊名称：JOURNAL OF VIROLOGICAL METHODS （影响因子：2.014；五年影响因子：2.001 ）

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收录情况： SCI

摘要： Two DNA constructs targeting the grass carp reovirus (GCRV) RNA dependent RNA polymerase (RdRp) gene and outer capsid protein (OCP) gene that were each 64 bp in length were synthesized chemically and cloned into pSilencer2.1-U6 neo plasmid, named pSi-RdRp1286 and pSi-OCP117, respectively. After transfection of pSi-RdRp 1286 and pSi-OCP117 plasmids into CIK cells, the inhibition of GCRV replication in the cells were detected by observing cytopathic effect (CPE), quantitating virus titers (TCID50/mL) and real-time quantitative RT-PCR analysis of viral RdRp and OCP genes. Five days after the cells were challenged with GCRV, both pSi-RdRp1286 and pSi-OCP117 reduced the viral titers by 5.47 IgTCID(50)/mL and 4.37 IgTCID(50)/mL, respectively. Compared to the positive control, CPE induced by GCRV in transfected cells was delayed and significantly less. Furthermore, the real-time quantitative RT-PCR analysis of the viral RdRp gene and OCP gene showed that the targeted gene expression were reduced by 89% and 73%, respectively. These results proved that the plasmid-transcribed shRNAs could inhibit effectively GCRV replication in CIK cells. These shRNAs provide potential tools for inhibiting GCRV infection and replication both in vitro and in vivo

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