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

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

摘要:

[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。

Abstract:

[Objective] To study the inhibitory effect of 3' untranslated region (UTR)-targeted artificial microRNA (amiRNA) 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 amiRNA 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 rAds, namely rAd-amiR3UTR-GFP and rAd-amiRcon-GFP, were generated. Both primary PAM and 3D4/163 cells could be transduced by rAd with different transduction efficiencies. The amiR3UTR was expressed in dose-and timedependent manners in rAd-transduced PAM cells. The amiR3UTR, but not amiRcon, had significant and stable inhibitory effects against replication of three different PRRSV strains in a dose-dependent manner. [Conclusion] The rAd-delivered amiR3UTR had strong anti-PRRSV effect against different PRRSV strains and rAd-amiR3UTR-GFP could be explored further as the alternative strategy against PRRS.