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构建一种多重靶向 RNAi 质粒,抑制 BHK-21 细胞和乳鼠中的靶基因表达和 FMDV 复制。

Construction of a multiple targeting RNAi plasmid that inhibits target gene expression and FMDV replication in BHK-21 cells and suckling mice.

机构信息

State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, 220 Handan Road, Shanghai, People's Republic of China.

出版信息

Vet Res Commun. 2010 Apr;34(4):335-46. doi: 10.1007/s11259-010-9360-y. Epub 2010 May 7.

Abstract

Foot-and-mouth disease (FMD) is a highly contagious disease that afflicts cloven-hoofed animals. The etiological agent of FMD is foot-and-mouth disease virus (FMDV). The VP1 gene of FMDV is essential during the life cycle of the virus and plays a key role in the attachment of the virus to susceptible cells. We constructed a plasmid, pCWN11, that expresses siRNAs multiple-targeting the VP1 genes of FMDV. We evaluated the gene silencing efficiency of the plasmid using an enhanced green fluorescent protein (EGFP) reporter system in BHK-21 cells. The antiviral potential of the plasmid in BHK-21 cells and suckling mice were investigated. The results indicate that cotransfection of pCWN11 with any one of three serotypes VP1-EGFP plasmids resulted in a reduction in the EGFP signal relative to the control. Moreover, the antiviral potential induced by pCWN11 was evident during challenge with one FMDV isolate of either serotype O (HKN/2002) or serotype Asia I (YNBS/58), and the inhibition extended to almost 40 h. Furthermore, subcutaneous injection of pCWN11 in the neck made suckling mice significantly less susceptible to FMDV serotype O and Asia I.

摘要

口蹄疫(FMD)是一种高度传染性疾病,影响偶蹄类动物。FMD 的病原体是口蹄疫病毒(FMDV)。FMDV 的 VP1 基因在病毒的生命周期中至关重要,在病毒附着于易感细胞中发挥关键作用。我们构建了一个表达针对 FMDV VP1 基因的多个 siRNA 的质粒 pCWN11。我们使用 BHK-21 细胞中的增强型绿色荧光蛋白(EGFP)报告系统评估了质粒的基因沉默效率。研究了质粒在 BHK-21 细胞和乳鼠中的抗病毒潜力。结果表明,pCWN11 与三种血清型 VP1-EGFP 质粒中的任何一种共转染都会导致 EGFP 信号相对于对照降低。此外,pCWN11 诱导的抗病毒潜力在与 O 型(HKN/2002)或亚洲 I 型(YNBS/58)的 FMDV 分离株的攻毒中显而易见,抑制作用持续近 40 小时。此外,颈部皮下注射 pCWN11 使乳鼠对 O 型和亚洲 I 型 FMDV 的易感性显著降低。

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