3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）

Inhibition of porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages with the 3′ UTR-targeted amiRNA

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。

[Objective] To study the inhibitory effect of 3′ untranslated region（UTR）-targeted artificial micro RNA（ami RNA） against porcine reproductive and respiratory syndrome virus（PRRSV） replication in porcine alveolar macrophages（PAM）. [Methods] Recombinant adenovirus（rAd） expressing the 3′ UTR-targeted or control amiRNA and green fluorescent protein（GFP） reporter gene were generated by transfecting AAV-293 cells with the transfer vector. The expression of sequence-specific ami RNA was detected by quantitative RT-PCR. The anti-PRRSV effect of amiRNA was detected by quantitative RT-PCR, Western blotting and viral titration assay. [Results] Two r Ads,namely r Ad-amiR3UTR-GFP and rAd-amiRcon-GFP, were generated. Both primary PAM and 3D4/163 cells could be transduced by r Ad with different transduction efficiencies. The amiR3UTR was expressed in dose-and timedependent manners in r Ad-transduced PAM cells. The amiR3UTR, but not ami Rcon, had significant and stable inhibitory effects against replication of three different PRRSV strains in a dose-dependent manner. [Conclusion] The r Ad-delivered amiR3UTR had strong anti-PRRSV effect against different PRRSV strains and r Ad-amiR3UTR-GFP could be explored further as the alternative strategy against PRRS.