多个发夹RNA的混合使用抑制O型和AsiaⅠ型口蹄疫病毒的复制

A Combination of Multiple Short Hairpin RNAs Inhibit the Replication of O and AsiaⅠType FMDV

通过对国内流行的口蹄疫病毒(FMDV)毒株的序列分析,锁定RNA干扰的3个目标基因即3D,VP4和2B,分别编码聚合酶POL,病毒结构蛋白VP4和非结构蛋白2B,这3个靶基因序列在不同流行毒株之间相对保守.分别设计合成靶向这些基因序列的发夹RNA(short-hairpin RNA,shRNA)表达模板,构建了5个shRNA表达质粒p3D-NT56,p3D-NT19,pVP4-NT65,pVP4-NT19和p2B-NT25.进行单独应用或混合应用,于细胞水平和乳鼠水平检测其抗病毒活性.结果显示,3D-NT56/19,VP4-NT65/19和2B-NT25 shRNA对O型FMDV均有抑制作用,但是对AsiaⅠ型FMDV,仅2B-NT25 shRNA具有较显著的抑制作用.3个shRNA表达质粒的混合使用(p3D-NT56+pVP4-NT65+p2B-NT25,p3D-NT19+pVP4-NT19+p2B-NT25),不仅能够交叉抑制不同血清型FMDV的复制,而且这种抑制作用较单一shRNA延续更长的时间.

Three RNAi targeted genes 3D, VP4 and 2B, which are conserved among different FMDV isolates of serotype O and serotype Asia I based on sequence analysis, were detemained. Short hairpin RNA （shRNA） -expressing templates were designed and synthesized, followed by cloning these shRNA-expressing templates into pCDNA3, 1-U6 plasmid, five shRNA-expressing plasmids p3D-NT56, p3D-NT19, pVP4-NT65, pVP4-NT19 and p2B-NT25 were constructed. The antiviral potentials of these shRNAs were investigated on BHK-21 cells and suckling mice, either by single or by a combination. The results showed that 3D-NT56/19, VP4-NT65/19 and 2B-NT25 shRNAs all have inhibition effects on serotype O FMDV （CHA/99）, but only 2B-NT25 shRNA showed a significant inhibition on serotype Asia I FMDV （YNBS/58）. However, the usage of three shRNA-expressing plasmids simultaneously gave a stable cross-inhibition to both isolates, and the antiviral effect extended longer than single shRNA.